Cytokine receptor zcytor17 multimers

ABSTRACT

Novel polypeptide combinations, polynucleotides encoding the polypeptides, and related compositions and methods are disclosed for zcytor17-containing multimeric or heterodimer cytokine receptors that may be used as novel cytokine antagonists, and within methods for detecting ligands that stimulate the proliferation and/or development of hematopoietic, lymphoid and myeloid cells in vitro and in vivo. The present invention also includes methods for producing the multimeric or heterodimeric cytokine receptor, uses therefor and antibodies thereto.

The present application is a divisional of U.S. patent application Ser.No. 10/351,157, filed Jan. 21, 2003, which claims the benefit of U.S.Patent Application Ser. No. 60/435,361, filed Dec. 19, 2002, U.S. PatentApplication Ser. No. 60/389,108, filed Jun. 14, 2002, and U.S. PatentApplication Ser. No. 60/350,325, filed Jan. 18, 2002, all of which areherein incorporated by reference.

BACKGROUND OF THE INVENTION

Proliferation and differentiation of cells of multicellular organismsare controlled by hormones and polypeptide growth factors. Thesediffusable molecules allow cells to communicate with each other and actin concert to form cells, tissues and organs, and to repair damagedtissue. Examples of hormones and growth factors include the steroidhormones (e.g., estrogen, testosterone), parathyroid hormone, folliclestimulating hormone, the interleukins, platelet derived growth factor(PDGF), epidermal growth factor (EGF), granulocyte-macrophage colonystimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.

Hormones and growth factors influence cellular metabolism by binding toreceptors. Receptors may be integral membrane proteins that are linkedto signaling pathways within the cell, such as second messenger systems.Other classes of receptors are soluble molecules, such as thetranscription factors.

Cytokines generally stimulate proliferation or differentiation of cellsof the hematopoietic lineage or participate in the immune andinflammatory response mechanisms of the body. Examples of cytokineswhich affect hematopoiesis are erythropoietin (EPO), which stimulatesthe development of red blood cells; thrombopoietin (TPO), whichstimulates development of cells of the megakaryocyte lineage; andgranulocyte-colony stimulating factor (G-CSF), which stimulatesdevelopment of neutrophils. These cytokines are useful in restoringnormal blood cell levels in patients suffering from anemia,thrombocytopenia, and neutropenia or receiving chemotherapy for cancer.

The interleukins are a family of cytokines that mediate immunologicalresponses, including inflammation. The interleukins mediate a variety ofinflammatory pathologies. Central to an immune response are T cells,which produce many cytokines and adaptive immunity to antigens.Cytokines produced by T cells have been classified as type 1 and type 2(Kelso, A. Immun. Cell Biol. 76:300-317, 1998). Type 1 cytokines includeIL-2, IFN-γ, LT-α, and are involved in inflammatory responses, viralimmunity, intracellular parasite immunity and allograft rejection. Type2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and are involvedin humoral responses, helminth immunity and allergic response. Sharedcytokines between Type 1 and 2 include IL-3, GM-CSF and TNF-α. There issome evidence to suggest that Type 1 and Type 2 producing T cellpopulations preferentially migrate into different types of inflamedtissue.

Mature T cells may be activated, i.e., by an antigen or other stimulus,to produce, for example, cytokines, biochemical signaling molecules, orreceptors that further influence the fate of the T cell population.

B cells can be activated via receptors on their cell surface including Bcell receptor and other accessory molecules to perform accessory cellfunctions, such as production of cytokines.

Monocytes/macrophages and T-cells can be activated by receptors on theircell surface and play a central role in the immune response bypresenting antigen to lymphocytes and also act as accessory cells tolymphocytes by secreting numerous cytokines.

Natural killer (K) cells have a common progenitor cell with T cells andB cells, and play a role in immune surveillance. NK cells, whichcomprise up to 15% of blood lymphocytes, do not express antigenreceptors, and therefore do not use MHC recognition as requirement forbinding to a target cell. NK cells are involved in the recognition andkilling of certain tumor cells and virally infected cells. In vivo, NKcells are believed to require activation, however, in vitro, NK cellshave been shown to kill some types of tumor cells without activation.

The demonstrated in vivo activities of these cytokines illustrate theenormous clinical potential of, and need for, other cytokines, cytokineagonists, and cytokine antagonists or binding partners. The presentinvention addresses these needs by providing a new hematopoieticmultimeric cytokine receptor, as well as related compositions andmethods.

The present invention provides such polypeptides for these and otheruses that should be apparent to those skilled in the art from theteachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a multiple alignment of human zcytor17lig(SEQ ID NO:2) (zcytor17lig), and mouse zcytor17lig (SEQ ID NO:11)(mzcytor17lig), mouse IL-3 (mIL-3) (SEQ ID NO:100), and human IL-3(hIL-3) (SEQ ID NO:102).

FIG. 2 is an illustration of a multiple alignment of human zcytor17lig(SEQ ID NO:2) (zcytor17lig), and mouse zcytor17lig (SEQ ID NO:11)(mzcytor17lig).

FIG. 3 is a Hopp/Woods hydrophilicity plot of human zcytor17lig (SEQ IDNO:2).

FIG. 4 is a multiple alignment of zcytor17 polynucleotide sequences SEQID NO:109, SEQ ID NO:113, SEQ ID NO:5, SEQ ID NO:111, and SEQ ID NO:115.

FIG. 5 is an alignment of human zcytor17 (ZCYTOR) (SEQ ID NO:5) andmouse zcytor17 (M17R—O) (SEQ ID NO:117). Between the two sequences,identical residues (:), Conserved residues (.) and gaps (−) areindicated.

SUMMARY OF THE INVENTION

The present invention provides an isolated multimeric or heterodimericcytokine receptor comprising at least one polypeptide having at least 90percent sequence identity with SEQ ID NO:111 or SEQ ID NO:109; andwherein the multimeric or heterodimeric cytokine receptor binds a ligandcomprising SEQ ID NO:2. Optionally, the isolated multimeric orheterodimeric cytokine receptor may further comprise a cytokine-bindingdomain of a class I cytokine receptor. The cytokine-binding domain ofthe class I cytokine receptor may comprise amino acid residue 28 toamino acid residue 429 of SEQ ID NO:7, amino acid residue 28 to aminoacid residue 739 of SEQ ID NO:7, amino acid residue 1 to amino acidresidue 429 of SEQ ID NO:7, amino acid residue 1 to amino acid residue739 of SEQ ID NO:7, amino acid residue 1 to amino acid residue 761 ofSEQ ID NO:7, amino acid residue 28 to amino acid residue 761 of SEQ IDNO:7, amino acid residue 28 to amino acid residue 979 of SEQ ID NO:7, oramino acid residue 1 to amino acid residue 979 of SEQ ID NO:7. Theisolated multimeric or heterodimeric cytokine receptor may antagonize anactivity of SEQ ID NO:2. The isolated multimeric or heterodimericcytokine receptor may inhibit proliferation of hematopoietic cells,inhibit proliferation of immune cells, inhibit proliferation ofinflammatory cells, inhibit an immune response, inhibit an inflammatoryresponse, or inhibit proliferation of tumor cells of epithelial origin.The isolated multimeric or heterodimeric cytokine receptor may besoluble. The isolated multimeric or heterodimeric cytokine receptor mayfurther comprises an affinity tag, such as, for instance, polyhistidine,protein A, glutathione S transferase, Glu-Glu, substance P, Flag™peptide, streptavidin binding peptide, and immunoglobulin F_(c)polypeptide, or cytotoxic molecule, such as, for instance, a toxin orradionuclide. The isolated multimeric or heterodimeric cytokine receptorwherein the polypeptide having at least 90 percent identity with SEQ IDNO:111 may comprise amino acid residue 20 to amino acid residue 227 ofSEQ ID NO:111, amino acid residue 20 to amino acid residue 519 of SEQ IDNO:111, amino acid residue 20 to amino acid residue 543 of SEQ IDNO:111, amino acid residue 20 to amino acid residue 732 of SEQ IDNO:111, amino acid residue 1 to amino acid residue 227, amino acidresidue 1 to amino acid residue 519, amino acid residue 1 to amino acidresidue 543, or amino acid residue 1 to amino acid residue 732. Theisolated multimeric or heterodimeric cytokine receptor wherein thepolypeptide having at least 90 percent identity with SEQ ID NO:109 maycomprise amino acid residue 1 to amino acid residue 649 of SEQ IDNO:109, or amino acid residue 20 to amino acid residue 649 of SEQ IDNO:109.

The present invention also provides an isolated multimeric orheterodimeric cytokine receptor comprising at least one polypeptidecomprising amino acid residue 20 to amino acid residue 227 of SEQ IDNO:111. The at least one polypeptide may comprise amino acid residue 1to amino acid residue 227 of SEQ ID NO:111, amino acid residue 20 toamino acid residue 519 of SEQ ID NO:111, amino acid residue 1 to aminoacid residue 519 of SEQ ID NO:111, amino acid residue 1 to amino acidresidue 543 of SEQ ID NO:111, amino acid residue 20 to amino acidresidue 543 of SEQ ID NO:111, amino acid residue 1 to amino acid residue732 of SEQ ID NO:111, or amino acid residue 20 to amino acid residue 732of SEQ ID NO:111. The isolated multimeric or heterodimeric cytokinereceptor may further comprise a cytokine-binding domain of a class Icytokine receptor, for instance, amino acid residue 28 to amino acidresidue 429 of SEQ ID NO:7, amino acid residue 1 to amino acid residue429 of SEQ ID NO:7, amino acid residue 28 to amino acid residue 739 ofSEQ ID NO:7, amino acid residue 1 to amino acid residue 739 of SEQ IDNO:7, amino acid residue 28 to amino acid residue 761 of SEQ ID NO:7,amino acid residue 1 to amino acid residue 761 of SEQ ID NO:7, aminoacid residue 28 to amino acid residue 979 of SEQ ID NO:7, or amino acidresidue 1 to amino acid residue 979 of SEQ ID NO:7. The isolatedmultimeric or heterodimeric cytokine receptor may antagonize an activityof a ligand comprising SEQ ID NO:2. The isolated multimeric orheterodimeric cytokine receptor may inhibit proliferation ofhematopoietic cells, inhibit proliferation of immune cells, inhibitproliferation of inflammatory cells, inhibit an immune response, inhibitan inflammatory response, or inhibit proliferation of tumor cells ofepithelial origin. Optionally, the isolated multimeric or heterodimericcytokine receptor may be is soluble. The isolated multimeric orheterodimeric cytokine receptor may further comprise an affinity tag,such as, for instance, polyhistidine, protein A, glutathione Stransferase, Glu-Glu, substance P, Flag™ peptide, streptavidin bindingpeptide, and immunoglobulin F_(c) polypeptide, or cytotoxic molecule,such as, for instance, a toxin or radionuclide.

The present invention also provides a soluble multimeric orheterodimeric cytokine receptor comprising amino acid residue 20 toamino acid residue 227 of SEQ ID NO:111 and amino acid residue 28 toamino acid residue 429 of SEQ ID NO:7.

The present invention also provides an isolated polynucleotide thatencodes a cytokine receptor polypeptide comprising an amino acidsequence having at least 90 percent sequence identity with SEQ ID NO:111or SEQ ID NO:109, wherein the cytokine receptor polypeptide forms amultimeric or heterodimeric cytokine receptor, and wherein themultimeric or heterodimeric cytokine receptor binds a ligand comprisingSEQ ID NO:2. The multimeric or heterodimeric cytokine receptor mayfurther comprise a cytokine-binding domain of a class I cytokinereceptor, such as, for instance, amino acid residue 28 to amino acidresidue 429 of SEQ ID NO:7, amino acid residue 28 to amino acid residue739 of SEQ ID NO:7, amino acid residue 1 to amino acid residue 429 ofSEQ ID NO:7, amino acid residue 1 to amino acid residue 739 of SEQ IDNO:7, amino acid residue 1 to amino acid residue 761 of SEQ ID NO:7,amino acid residue 28 to amino acid residue 761 of SEQ ID NO:7, aminoacid residue 28 to amino acid residue 979 of SEQ ID NO:7, or amino acidresidue 1 to amino acid residue 979 of SEQ ID NO:7. The multimeric orheterodimeric cytokine receptor may antagonize an activity of SEQ IDNO:2. The multimeric or heterodimeric cytokine receptor may inhibitproliferation of hematopoietic cells, inhibit proliferation of immunecells, inhibit proliferation of inflammatory cells, inhibit an immuneresponse, inhibit an inflammatory response, or inhibit proliferation oftumor cells of epithelial origin. Optionally, the multimeric orheterodimeric cytokine receptor may be soluble. The multimeric orheterodimeric cytokine receptor may further comprise an affinity tag,such as, for instance, polyhistidine, protein A, glutathione Stransferase, Glu-Glu, substance P, Flag™ peptide, streptavidin bindingpeptide, and immunoglobulin F_(c) polypeptide, or cytotoxic molecule,such as, for instance, a toxin or radionuclide. The encoded cytokinereceptor polypeptide having at least 90 percent identity with SEQ IDNO:111 may comprise amino acid residue 20 to amino acid residue 227 ofSEQ ID NO:111, amino acid residue 20 to amino acid residue 519 of SEQ IDNO:111, amino acid residue 20 to amino acid residue 543 of SEQ IDNO:111, amino acid residue 20 to amino acid residue 732 of SEQ IDNO:111, amino acid residue 1 to amino acid residue 227 of SEQ ID NO:111,amino acid residue 1 to amino acid residue 519 of SEQ ID NO:111, aminoacid residue 1 to amino acid residue 543 of SEQ ID NO:111, or amino acidresidue 1 to amino acid residue 732 of SEQ ID NO:111. The encodedcytokine receptor polypeptide having at least 90 percent identity withSEQ ID NO:109 may comprise amino acid residue 1 to amino acid residue649 of SEQ ID NO:109, or amino acid residue 20 to amino acid residue 649of SEQ ID NO:109.

The present invention also provides an isolated polynucleotide thatencodes a cytokine receptor polypeptide comprising amino acid residue 20to amino acid residue 227 of SEQ ID NO:111, wherein the cytokinereceptor polypeptide forms a multimeric or heterodimeric cytokinereceptor. The cytokine receptor polypeptide may comprise amino acidresidue 1 to amino acid residue 227 of SEQ ID NO:111, amino acid residue20 to amino acid residue 519 of SEQ ID NO:111, amino acid residue 1 toamino acid residue 519 of SEQ ID NO:111, amino acid residue 1 to aminoacid residue 543 of SEQ ID NO:111, amino acid residue 20 to amino acidresidue 543 of SEQ ID NO:111, amino acid residue 1 to amino acid residue732 of SEQ ID NO:111, or amino acid residue 20 to amino acid residue 732of SEQ ID NO:111. The multimeric or heterodimeric cytokine receptor mayfurther comprise a cytokine-binding domain of a class I cytokinereceptor, such as, for instance, amino acid residue 28 to amino acidresidue 429 of SEQ ID NO:7, amino acid residue 1 to amino acid residue429 of SEQ ID NO:7, amino acid residue 28 to amino acid residue 739 ofSEQ ID NO:7, amino acid residue 1 to amino acid residue 739 of SEQ IDNO:7, amino acid residue 28 to amino acid residue 761 of SEQ ID NO:7,amino acid residue 1 to amino acid residue 761 of SEQ ID NO:7, aminoacid residue 28 to amino acid residue 979 of SEQ ID NO:7, or amino acidresidue 1 to amino acid residue 979 of SEQ ID NO:7. The multimeric orheterodimeric cytokine receptor may antagonize an activity of a ligandcomprising SEQ ID NO:2. The multimeric or heterodimeric cytokinereceptor may inhibit proliferation of hematopoietic cells, inhibitproliferation of immune cells, inhibit proliferation of inflammatorycells, inhibit an immune response, inhibit an inflammatory response, orinhibit proliferation of tumor cells of epithelial origin. Optionally,the multimeric or heterodimeric cytokine receptor may be soluble. Themultimeric or heterodimeric cytokine receptor may further comprise anaffinity tag or cytotoxic molecule as described herein.

The present invention also provides an expression vector that comprisesthe the following operably linked elements: a transcription promoter; aDNA segment encoding a cytokine receptor polypeptide having at least 90percent sequence identity with SEQ ID NO:111; and a transcriptionterminator; wherein the cytokine receptor polypeptide forms a multimericor heterodimeric cytokine receptor, and wherein the multimeric orheterodimeric cytokine receptor binds a ligand comprising SEQ ID NO:2.

Alternatively, the present invention also provides an expression vectorthat comprises the following operably linked elements: a) a firsttranscription promoter; a first DNA segment encoding a cytokine receptorpolypeptide having at least 90 percent sequence identity with SEQ IDNO:111; and a first transcription terminator; and b) a secondtranscription promoter; a second DNA segment encoding a cytokine-bindingdomain of a class I cytokine receptor; and a second transcriptionterminator; wherein the cytokine receptor polypeptide and the class Icytokine receptor form a multimeric or heterodimeric cytokine receptor;and wherein the multimeric or heterodimeric cytokine receptor binds to aligand comprising SEQ ID NO:2.

Alternatively, the present invention also provides an expression vectorthat comprises the following operably linked elements: a) a firsttranscription promoter; a first DNA segment encoding a polypeptidehaving at least 90 percent sequence identity with SEQ ID NO:111; and afirst transcription terminator; and b) a second transcription promoter;a second DNA segment encoding at least a portion of a class I cytokinereceptor; and a second transcription terminator; wherein the polypeptideand the class I cytokine receptor form a multimeric cytokine receptor;and wherein the multimeric cytokine receptor binds to at least a portionof SEQ ID NO:2.

The expression vectors of the present invention may further include asecretory signal sequence linked to the first and second DNA segments.The multimeric or heterodimeric cytokine receptor may be soluble,membrane-bound, or attached to a solid support. The multimeric orheterodimeric cytokine receptor may antagonize an activity of a ligandcomprising SEQ ID NO:2. The multimeric or heterodimeric cytokinereceptor may inhibit proliferation of hematopoietic cells, inhibitproliferation of immune cells, inhibit proliferation of inflammatorycells, inhibit an immune response, inhibit an inflammatory response, orinhibit proliferation of tumor cells of epithelial origin. Optionally,the multimeric or heterodimeric cytokine receptor may be soluble. Themultimeric or heterodimeric cytokine receptor may further comprise anaffinity tag or cytotoxic molecule as described herein.

The present invention also provides a cultured cell including anexpression vector as discribed herein, wherein the cell expresses thepolypeptide or polypeptides encoded by the DNA segment or segments. Thecell may secrete the multimeric or heterodimeric cytokine receptor. Themultimeric cytokine receptor may bind and/or antagonize an activity ofSEQ ID NO:2 as further described herein.

The present invention also provides a cultured cell which includes afirst expression vector comprising: a) a transcription promoter; b) aDNA segment encoding a cytokine receptor polypeptide having at least 90percent sequence identity with SEQ ID NO:111; and c) a transcriptionterminator; and a second expression vector comprising: a) atranscription promoter; b) a DNA segment encoding a cytokine-bindingdomain of a class I cytokine receptor; and c) a transcriptionterminator; wherein the cytokine receptor polypeptide and the class Icytokine receptor form a multimeric or heterodimeric cytokine receptor,and wherein the multimeric or heterodimeric cytokine receptor binds to aligand that comprises SEQ ID NO:2. The first and second expressionvectors may include a secretory signal sequence operably linked to thefirst and second DNA segments. The cultured cell may further comprise athird expression vector which includes a) a transcription promoter; b) aDNA segment encoding a cytokine-binding domain of a second class Icytokine receptor; and c) a transcription terminator; wherein thecytokine receptor polypeptide, the first class I cytokine receptor, andthe second class I cytokine receptor form a multimeric cytokinereceptor. The cytokine-binding domain of a class I cytokine receptor maybe of SEQ ID NO:7 and/or SEQ ID NO:9. Optionally, the multimeric orheterodimeric cytokine receptor may be soluble. The multimeric orheterodimeric cytokine receptor may further include an affinity tag asdescribed herein. The multimeric or heterodimeric cytokine receptor maybind to at least a portion of SEQ ID NO:2 and/or antagonize an activityof SEQ ID NO:2 as described herein.

The present invention also provides a method of producing an antibody toa multimeric or heterodimeric cytokine receptor comprising amino acidresidue 20 to amino acid residue 227 of SEQ ID NO:111 and acytokine-binding domain of a class I cytokine receptor. The methodincludes inoculating an animal with the multimeric or heterodimericcytokine receptor, wherein the multimeric or heterodimeric cytokinereceptor elicits an immune response in the animal to produce an antibodythat specifically binds the multimeric or heterodimeric cytokinereceptor; and isolating the antibody from the animal. The antibody mayoptionally be a monoclonal antibody. The antibody may optionally be aneutralizing antibody. The antibody may specifically bind to amultimeric or heterodimeric cytokine receptor as described herein.

The present invention also provides a composition which includes aneffective amount of a soluble multimeric or heterodimeric cytokinereceptor comprising amino acid residue 20 to amino acid residue 227 ofSEQ ID NO:111 and a cytokine-binding domain of a class I cytokinereceptor; and a pharmaceutically acceptable vehicle. The binding domainof the class I cytokine receptor may include amino acid residue 28 toamino acid residue 429 of SEQ ID NO:7. The soluble multimeric orheterodimeric cytokine receptor may bind to a ligand comprising SEQ IDNO:2. The soluble multimeric or heterodimeric cytokine receptor mayfurther include an affinity tag or cytotoxic molecule as describedherein. The composition may antagonize an activity of a ligandcomprising SEQ ID NO:2. The composition may inhibit proliferation ofhematopoietic cells, inhibit proliferation of immune cells, inhibitproliferation of inflammatory cells, inhibit an immune response, inhibitan inflammatory response, or inhibit proliferation of tumor cells ofepithelial origin

The present invention also provides a method of producing a multimericor heterodimeric cytokine receptor comprising culturing a cell asdescribed herein, and isolating the multimeric or heterodimeric cytokinereceptor produced by the cell.

The present invention also provides an immune cell inhibitingcomposition which includes an effective amount of a soluble multimericor heterodimeric cytokine receptor comprising amino acid residue 20 toamino acid residue 227 of SEQ ID NO:111 and a cytokine-binding domain ofa class I cytokine receptor; and a pharmaceutically acceptable vehicle;wherein the soluble multimeric or heterodimeric cytokine receptorinhibits the proliferation of immune cells.

The present invention also provides an immune response inhibitingcomposition which includes an effective amount of a soluble multimericor heterodimeric cytokine receptor comprising amino acid residue 20 toamino acid residue 227 of SEQ ID NO:111 and a cytokine-binding domain ofa class I cytokine receptor; and a pharmaceutically acceptable vehicle;wherein the soluble multimeric or heterodimeric cytokine receptorinhibits an immune response.

The present invention also provides an inflammatory cell inhibitingcomposition which includes an effective amount of a soluble multimericor heterodimeric cytokine receptor comprising amino acid residue 20 toamino acid residue 227 of SEQ ID NO:111 and a cytokine-binding domain ofa class I cytokine receptor; and a pharmaceutically acceptable vehicle;wherein the soluble multimeric or heterodimeric cytokine receptorinhibits the proliferation of inflammatory cells.

The present invention also provides an inflammatory response inhibitingcomposition which includes an effective amount of a soluble multimericor heterodimeric cytokine receptor comprising amino acid residue 20 toamino acid residue 227 of SEQ ID NO:111 and a cytokine-binding domain ofa class I cytokine receptor; and a pharmaceutically acceptable vehicle;wherein the soluble multimeric or heterodimeric cytokine receptorinhibits an inflammatory response.

The present invention also provides a method of inhibiting an immuneresponse in a mammal exposed to an antigen or pathogen. The methodincludes (a) determining directly or indirectly the level of antigen orpathogen present in the mammal; (b) administering a compositioncomprising a soluble multimeric or heterodimeric cytokine receptor in apharmaceutically acceptable vehicle; (c) determining directly orindirectly the level of antigen or pathogen in the mammal; and (d)comparing the level of the antigen or pathogen in step (a) to theantigen or pathogen level in step (c), wherein a change in the level isindicative of inhibiting an immune response. The method may furthercomprise (e) re-administering a composition comprising a multimericcytokine receptor in a pharmaceutically acceptable vehicle; (f)determining directly or indirectly the level of antigen or pathogen inthe mammal; and (g) comparing the number of the antigen or pathogenlevel in step (a) to the antigen level in step (f), wherein a change inthe level is indicative of inhibiting an immune response.

The present invention also provides a method for reducing hematopoieticcells and/or hematopoietic progenitors cells in a mammal. The methodincludes culturing bone marrow or peripheral blood cells with acomposition comprising an effective amount of a soluble multimeric orheterodimeric cytokine receptor to produce a decrease in the number oflymphoid cells in the bone marrow or peripheral blood cells as comparedto bone marrow or peripheral blood cells cultured in the absence of themultimeric cytokine receptor. The hematopoietic cells and hematopoieticcell progenitors may be lymphoid, which can be monocytic cells,macrophages, or T cells.

The present invention also provides a method of detecting the presenceof a multimeric or heterodimeric cytokine receptor in a biologicalsample. The method includes contacting the biological sample with anantibody, or an antibody fragment, as described herein, wherein thecontacting is performed under conditions that allow the binding of theantibody or antibody fragment to the biological sample; and detectingany of the bound antibody or bound antibody fragment.

The present invention also provides a method of a method of killingcancer cells. The method includes obtaining ex vivo a tissue orbiological sample containing cancer cells from a patient, or identifyingcancer cells in vivo; producing a multimeric or heterodimeric cytokinereceptor by a method as described herein; formulating the multimeric orheterodimeric cytokine receptor in a pharmaceutically acceptablevehicle; and administering to the patient or exposing the cancer cellsto the multimeric or heterodimeric cytokine receptor formulation;wherein the multimeric or heterodimeric cytokine receptor kills thecells. The multimeric or heterodimeric cytokine receptor may be furtherconjugated to a toxin.

The present invention also provides an antibody that specifically bindsto a multimerc or heterodimeric cytokine receptor as described herein.The antibody may be a polyclonal antibody, a murine monoclonal antibody,a humanized antibody derived from a murine monoclonal antibody, anantibody fragment, a neutralizing antibody, or a human monoclonalantibody. The antibody or antibody fragment may specifically bind to amultimeric or heterodimeric cytokine receptor of the present inventionwhich may comprise a cytokine receptor polypeptide comprising amino acidresidue 20 to amino acid residue 227 of SEQ ID NO:111 and acytokine-binding domain of a class I cytokine receptor. The antibody mayfurther include a radionuclide, enzyme, substrate, cofactor, fluorescentmarker, chemiluminescent marker, peptide tag, magnetic particle, drug,or toxin.

The present invention also provides a method for inhibitingzcytor17lig-induced proliferation or differentiation of hematopoieticcells and hematopoietic progenitor cells. The method includes culturingbone marrow or peripheral blood cells with a composition comprising anamount of a soluble multimeric or heterodimeric cytokine receptorcomprising a cytokine receptor polypeptide comprising amino acid residue20 to amino acid residue 227 of SEQ ID NO:111 and a cytokine-bindingdomain of a class I cytokine receptor sufficient to reduce proliferationor differentiation of the hematopoietic cells in the bone marrow orperipheral blood cells as compared to bone marrow or peripheral bloodcells cultured in the absence of the soluble multimeric or heterodimericcytokine receptor. The hematopoietic cells and hematopoietic progenitorcells may be lymphoid cells, such as macrophages or T cells.

The present invention also provides a method of reducingzcytor17lig-induced induced inflammation. The method includesadministering to a mammal with inflammation an amount of a compositioncomprising amino acid residue 20 to amino acid residue 227 of SEQ IDNO:111 and a cytokine-binding domain of a class I cytokine receptorsufficient to reduce inflammation.

The present invention also provides a method of suppressing aninflammatory response in a mammal with inflammation. The method includes(1) determining a level of an inflammatory molecule; (2) administering acomposition comprising amino acid residue 20 to amino acid residue 227of SEQ ID NO:111 and a cytokine-binding domain of a class I cytokinereceptor in a pharmaceutically acceptable vehicle; (3) determining apost administration level of the inflammatory molecule; (4) comparingthe level of the inflammatory molecule in step (1) to the level of theinflammatory molecule in step (3), wherein a lack of increase or adecrease the inflammatory molecule level is indicative of suppressing aninflammatory response.

The present invention also provides a method for inhibitingzcytor17lig-induced proliferation or differentiation of hematopoieticcells and hematopoietic progenitor cells. The method includes culturingbone marrow or peripheral blood cells with a composition comprisingamino acid residue 20 to amino acid residue 227 of SEQ ID NO:111 and acytokine-binding domain of a class I cytokine receptor in apharmaceutically acceptable vehicle sufficient to reduce proliferationor differentiation of the hematopoietic cells in the bone marrow orperipheral blood cells as compared to bone marrow or peripheral bloodcells cultured in the absence of soluble multimeric or heterodimericcytokine receptor. The hematopoietic cells and hematopoietic progenitorcells may be lymphoid cells, such as macrophages or T cells.

The present invention also provides a method of reducingzcytor17lig-induced induced inflammation. The method includesadministering to a mammal with inflammation an amount of a compositioncomprising amino acid residue 20 to amino acid residue 227 of SEQ IDNO:111 and a cytokine-binding domain of a class I cytokine receptor in apharmaceutically acceptable vehicle sufficient to reduce inflammation.

The present invention also provides a method of suppressing aninflammatory response in a mammal with inflammation. The method includes(1) determining a level of an inflammatory molecule; (2) administering acomposition comprising a multimeric or heterodimeric cytokine receptorwhich comprises amino acid residue 20 to amino acid residue 227 of SEQID NO:111 in a pharmaceutically acceptable vehicle; (3) determining apost administration level of the inflammatory molecule; (4) comparingthe level of the inflammatory molecule in step (1) to the level of theinflammatory molecule in step (3), wherein a lack of increase or adecrease in the inflammatory molecule level is indicative of suppressingan inflammatory response.

The present invention also provides a method of treating a mammalafflicted with an inflammatory disease in which zcytor17lig plays arole. The method includes administering an antagonist of zcytor17lig tothe mammal such that the inflammation is reduced, wherein the antagonistis a soluble multimeric or heterodimeric cytokine receptor comprisingamino acid residue 20 to amino acid residue 227 of SEQ ID NO:111 and acytokine-binding domain of a class I cytokine receptor in apharmaceutically acceptable vehicle. The inflammatory disease may be achronic inflammatory disease, such as, for instance, inflammatory boweldisease, ulcerative colitis, Crohn's disease, atopic dermatitis, eczema,or psoriasis. The inflammatory disease may be an acute inflammatorydisease, such as, for instance, endotoxemia, septicemia, toxic shocksyndrome, or infectious disease. Optinally, the soluble multimeric orheterodimeric cytokine receptor may further comprise a radionuclide,enzyme, substrate, cofactor, fluorescent marker, chemiluminescentmarker, peptide tag, magnetic particle, drug, or toxin.

The present invention also provides a method for detecting inflammationin a patient. The method includes obtaining a tissue or biologicalsample from a patient; incubating the tissue or biological sample with asoluble multimeric or heterodimeric cytokine receptor comprising aminoacid residue 20 to amino acid residue 227 of SEQ ID NO:111 and acytokine-binding domain of a class I cytokine receptor under conditionswherein the soluble multimeric or heterodimeric cytokine receptor bindsto its complementary polypeptide in the tissue or biological sample;visualizing the soluble multimeric or heterodimeric cytokine receptorbound in the tissue or biological sample; and comparing levels ofsoluble multimeric or heterodimeric cytokine receptor bound in thetissue or biological sample from the patient to a normal control tissueor biological sample, wherein an increase in the level of solublemultimeric or heterodimeric cytokine receptor bound to the patienttissue or biological sample relative to the normal control tissue orbiological sample is indicative of inflammation in the patient.

The present invention also provides a method for detecting a multiplecytokine receptor ligand from a test sample. The method includescontacting the test sample with a multimeric or heterodimeric cytokinereceptor comprising a cytokine receptor polypeptide comprising aminoacid residue 20 to amino acid residue 227 of SEQ ID NO:111 and acytokine-binding domain of a class I cytokine receptor; and detectingthe binding of the multimeric or heterodimeric cytokine receptor to theligand in the test sample.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” denotes apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a polynucleotide that has a contiguous stretchof identical or complementary sequence to another polynucleotide.Contiguous sequences are said to “overlap” a given stretch ofpolynucleotide sequence either in their entirety or along a partialstretch of the polynucleotide. For example, representative contigs tothe polynucleotide sequence 5′-ATGGCTTAGCTT-3′ are 5′-TAGCTTgagtct-3′and 3′-gtcgacTACCGA-5′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “neoplastic”, when referring to cells, indicates cellsundergoing new and abnormal proliferation, particularly in a tissuewhere in the proliferation is uncontrolled and progressive, resulting ina neoplasm. The neoplastic cells can be either malignant, i.e., invasiveand metastatic, or benign.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis. Soluble receptor polypeptides are said to be substantiallyfree of transmembrane and intracellular polypeptide segments when theylack sufficient portions of these segments to provide membrane anchoringor signal transduction, respectively.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

The present invention is based in part upon the discovery of a novelmultimeric cytokine receptor protein having the structure of a class Icytokine receptor, referred to herein as “multimeric cytokine receptor,”or “zcytor17 multimeric cytokine receptor.” The multimeric cytokinereceptor includes at least a portion of a zcytor17 receptor subunit,disclosed in the commonly owned U.S. patent spplication Ser. No.09/892,949. Another receptor subunit polypeptide that may be included inthe multimeric cytokine receptor of the present invention includes atleast a portion of at least one polypeptide of a class I cytokinereceptor, such as OSMRbeta and/or WSX-1. For example, the deduced aminoacid sequence indicated that zcytor17 belongs to the receptor subfamilythat includes gp130, LIF, IL-12, oncostatinM receptor beta (OSMRbeta)(SEQ ID NO:7), WSX-1 receptors (SEQ ID NO:9) (Sprecher, C A et al.,Biochem. Biophys. Res. Comm., 246:81-90 (1998); and U.S. Pat. No.5,925,735), DCRS2 (WIPO Publication No. WO 00/73451), the IL-2 receptorβ-subunit and the β-common receptor (i.e., IL-3, IL-5, and GM-CSFreceptor subunits). A further example of class I cytokine receptorsubunit polypeptides that may be included in the multimeric cytokinereceptor are the receptors for IL-2, IL-4, IL-7, Lif, IL-12, IL-15, EPO,TPO, GM-CSF and G-CSF (Cosman, Cytokine, 5(2):95-106 (1993)).

Cytokine receptor subunits are characterized by a multi-domain structurecomprising an extracellular domain, a transmembrane domain that anchorsthe polypeptide in the cell membrane, and an intracellular domain. Theextracellular domain may be a ligand-binding domain, and theintracellular domain may be an effector domain involved in signaltransduction, although ligand-binding and effector functions may resideon separate subunits of a multimeric receptor. The ligand-binding domainmay itself be a multi-domain structure. Multimeric receptors includehomodimers (e.g., PDGF receptor αα and ββ isoforms, erythropoietinreceptor, MPL, and G-CSF receptor), heterodimers whose subunits eachhave ligand-binding and effector domains (e.g., PDGF receptor αβisoform), and multimers having component subunits with disparatefunctions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSFreceptors). Some receptor subunits are common to a plurality ofreceptors. For example, the AIC2B subunit, which cannot bind ligand onits own but includes an intracellular signal transduction domain, is acomponent of IL-3 and GM-CSF receptors. Many cytokine receptors can beplaced into one of four related families on the basis of the structureand function. Hematopoietic receptors, for example, are characterized bythe presence of a domain containing conserved cysteine residues and theWSXWS motif (SEQ ID NO:3). Cytokine receptor structure has been reviewedby Urdal, Ann. Reports Med. Chem. 26:221-228, 1991 and Cosman, Cytokine5:95-106, 1993. Under selective pressure for organisms to acquire newbiological functions, new receptor family members likely arise fromduplication of existing receptor genes leading to the existence ofmulti-gene families. Family members thus contain vestiges of theancestral gene, and these characteristic features can be exploited inthe isolation and identification of additional family members. Thus, thecytokine receptor superfamily is subdivided into several families, forexample, the immunoglobulin family (including CSF-1, MGF, IL-1, and PDGFreceptors); the hematopoietin family (including IL-2 receptor β-subunit,GM-CSF receptor a-subunit, GM-CSF receptor β-subunit; and G-CSF, EPO,IL-3, IL-4, IL-5, IL-6, IL-7, and IL-9 receptors); TNF receptor family(including TNF (p80) TNF (p60) receptors, CD27, CD30, CD40, Fas, and NGFreceptor).

Analysis of the zcytor17 sequence suggests that it is a member of thesame receptor subfamily as the gp130, LIF, IL-12, WSX-1, IL-2 receptorβ-subunit, IL-3, IL-4, and IL-6 receptors. Certain receptors in thissubfamily (e.g., G-CSF) associate to form homodimers that transduce asignal. Other members of the subfamily (e.g., gp130, IL-6, IL-11, andLIF receptors) combine with a second subunit (termed a β-subunit) tobind ligand and transduce a signal. Specific β-subunits associate with aplurality of specific cytokine receptor subunits. For example, theβ-subunit gp130 (Hibi et al., Cell 63:1149-1157, 1990) associates withreceptor subunits specific for IL-6, IL-11, and LIF (Gearing et al.,EMBO J. 10:2839-2848, 1991; Gearing et al., U.S. Pat. No. 5,284,755).Oncostatin M binds to a heterodimer of LIF receptor and gp130. CNTFbinds to trimeric receptors comprising CNTF receptor, LIF receptor, andgp130 subunits.

A multimeric cytokine receptor of the present invention can be aheterodimer, trimer, tetramer, pentamer, and the like, comprising atleast a portion of zcytor17 and at least a portion of a class I cytokinereceptor. In addition, a multimeric cytokine receptor can be soluble,membrane-bound, or attached to a solid support. Analysis of the tissuedistribution of the mRNA of the zcytor17 receptor revealed expression inactivated CD4+ and CD8+ T-cell subsets, CD14+ monocytes, and weakerexpression in CD19+ B-cells. Moreover, the mRNA was present in bothresting or activated monocytic cell lines THP-1 (ATCC No. TIB-202), U937(ATCC No. CRL-1593.2) and HL60 (ATCC No. CCL-240).

Nucleotide sequences of representative zcytor17-encoding DNA aredescribed in SEQ ID NO:110 (from nucleotide 171 to 2366), with itsdeduced 732 amino acid sequence described in SEQ ID NO:111; SEQ IDNO:108 (from nucleotide 162 to 2108), with its deduced 649 amino acidsequence described in SEQ ID NO:109; and in SEQ ID NO:4 (from nucleotide497 to 2482), with its deduced 662 amino acid sequence described in SEQID NO:5. In its entirety, the zcytor17 polypeptide (SEQ ID NO:111, SEQID NO:109 or SEQ ID NO:5) represents a full-length polypeptide segment(residue 1 (Met) to residue 732 (Val) of SEQ ID NO:111; residue 1 (Met)to residue 649 (Ile) of SEQ ID NO:109; residue 1 (Met) to residue 662(Ile) of SEQ ID NO:5). The domains and structural features of thezcytor17 polypeptides are further described below.

Analysis of the zcytor17 polypeptide encoded by the DNA sequence of SEQID NO:110 revealed an open reading frame encoding 732 amino acids (SEQID NO:111) comprising a predicted secretory signal peptide of 19 aminoacid residues (residue 1 (Met) to residue 19 (Ala) of SEQ ID NO:111),and a mature polypeptide of 713 amino acids (residue 20 (Ala) to residue732 (Val) of SEQ ID NO:111). Analysis of the zcytor17 polypeptideencoded by the DNA sequence of SEQ ID NO:108 revealed an open readingframe encoding 649 amino acids (SEQ ID NO:109) comprising a predictedsecretory signal peptide of 19 amino acid residues (residue 1 (Met) toresidue 19 (Ala) of SEQ ID NO:109), and a mature polypeptide of 630amino acids (residue 20 (Ala) to residue 649 (Ile) of SEQ ID NO:109).Analysis of the zcytorl 7 polypeptide encoded by the DNA sequence of SEQID NO:4 revealed an open reading frame encoding 662 amino acids (SEQ IDNO:5) comprising a predicted secretory signal peptide of 32 amino acidresidues (residue 1 (Met) to residue 32 (Ala) of SEQ ID NO:5), and amature polypeptide of 630 amino acids (residue 33 (Ala) to residue 662(Ile) of SEQ ID NO:5). In addition to the WSXWS motif (SEQ ID NO:3)(corresponding to residues 211 to 215 of SEQ ID NO:111 and SEQ IDNO:109; and residues 224 to 228 of SEQ ID NO:5), the receptor comprisesan extracellular domain (residues 20 (Ala) to 519 (Glu) of SEQ ID NO:111and SEQ ID NO:109; residues 33 (Ala) to 532 (Glu) of SEQ ID NO:5) whichincludes a cytokine-binding domain of approximately 200 amino acidresidues (residues 20 (Ala) to 227 (Pro) of SEQ ID NO:111 and SEQ IDNO:109; residues 33 (Ala) to 240 (Pro) of SEQ ID NO:5); a domain linker(residues 122 (Thr) to 125 (Pro) of SEQ ID NO:111 and SEQ ID NO:109;residues 135 (Thr) to 138 (Pro) of SEQ ID NO:111); a penultimate strandregion (residues 194 (Phe) to 202 (Arg) of SEQ ID NO:111 and SEQ IDNO:109; residues 207 (Phe) to 215 (Arg) of SEQ ID NO:5); a fibronectintype III domain (residues 228 (Cys) to 519 (Glu) of SEQ ID NO:111 andSEQ ID NO:109; residues 241 (Cys) to 532 (Glu) of SEQ ID NO:5); atransmembrane domain (residues 520 (Ile) to 543 (Leu) of SEQ ID NO:111and SEQ ID NO:109; residues 533 (Ile) to 556 (Leu) of SEQ ID NO:5);complete intracellular signaling domain (residues 544 (Lys) to 732 (Val)of SEQ ID NO:111; residues 544 (Lys) to 649 (Ile) of SEQ ID NO:109; andresidues 557 (Lys) to 662 (Ile) of SEQ ID NO:5) which contains a “Box I”signaling site (residues 554 (Trp) to 560 (Pro) of SEQ ID NO:111 and SEQID NO:109; residues 567 (Trp) to 573 (Pro) of SEQ ID NO:5), and a “BoxII” signaling site (residues 617 (Gln) to 620 (Phe) of SEQ ID NO:111 andSEQ ID NO:109; residues 630 (Gln) to 633 (Phe) of SEQ ID NO:5). Thoseskilled in the art will recognize that these domain boundaries areapproximate, and are based on alignments with known proteins andpredictions of protein folding. In addition to these domains, conservedreceptor features in the encoded receptor include (as shown in SEQ IDNO:111 and SEQ ID NO:109) a conserved Cys residue at position 30(position 43 as shown in SEQ ID NO:5), CXW motif (wherein X is any aminoacid) at positions 40-42 (positions 53-55 as shown in SEQ ID NO:5), Trpresidue at position 170 (position 183 as shown in SEQ ID NO:5), and aconserved Arg residue at position 202 (position 215 as shown in SEQ IDNO:5). The corresponding polynucleotides encoding the zcytor17polypeptide regions, domains, motifs, residues and sequences describedabove are as shown in SEQ ID NO:110, SEQ ID NO:108, and SEQ ID NO:4.

Moreover, truncated forms of the zcytor17 polypeptide appear to benaturally expressed. Both forms encode soluble zcytor17 receptors. Apolynucleotide encoding a “long-form” of the soluble zcytor17 receptor,truncated within the fibronectin type III domain, is shown in SEQ IDNO:112 and the corresponding polypeptide is shown in SEQ ID NO:113. Thistruncated form encodes residues 1 (Met) through 324 (Lys) of SEQ IDNO:111 and SEQ ID NO:109), and thus comprises an intact signal sequence,WSXWS (SEQ ID NO:3) motif, linker, cytokine binding domain, penultimatestrand, and conserved, Cys, CXW motif, Trp and Arg residues as describedabove. A polynucleotide encoding a “short-form” of the soluble zcytor17receptor, truncated at the end of the cytokine binding domain is shownin SEQ ID NO:114 and the corresponding polypeptide is shown in SEQ IDNO:115. This truncated form encodes a 239 residue polypeptide that isidentical to residues 1 (Met) through 225 (Glu) of SEQ ID NO:111 and SEQID NO:109 and then diverges, and thus comprises an intact signalsequence, WSXWS (SEQ ID NO:3) motif, linker, cytokine binding domain,penultimate strand, and conserved, Cys, CXW motif, Trp and Arg residuesas described above. A multiple alignment of the truncated forms comparedto the full-length forms of zcytor17 is shown in FIG. 1.

Moreover, the zcytor17 cDNA of SEQ ID NO:110, SEQ ID NO:108, SEQ IDNO:112, and SEQ ID NO:114 encode polypeptides that may use analternative initiating methionine (at nucleotide 75 of SEQ ID NO:110, atnucleotide 66 of SEQ ID NO:108, at nucleotide 66 of SEQ ID NO:112, andat nucleotide 66 of SEQ ID NO:114) that would encode a polypeptide inthe same open reading frame (ORF) as the zcytor17 polypeptides of SEQ IDNO:111, SEQ ID NO:109, SEQ ID NO:113, and SEQ ID NO:115. Use of thealternative initiating methionine would add 32 amino acids (shown in SEQID NO:48) in-frame to the N-terminus of SEQ ID NO:111, SEQ ID NO:109,SEQ ID NO:113, and SEQ ID NO:111. In addition, nucleotide 536 of SEQ IDNO:4 may serve as an alternative initiating methionine, thus generatingthe same N-terminus (starting at amino acid 14 (Met) of SEQ ID NO:5) andsignal polypeptide sequence, as SEQ ID NO:111, SEQ ID NO:109, SEQ IDNO:113, and SEQ ID NO:115. Moreover, the second Met at amino acid number2 in the SEQ ID NO:111, SEQ ID NO:109, SEQ ID NO:113, and SEQ ID NO:115sequences (similarly at amino acid number 15 (Met) in SEQ ID NO:5) mayalso serve as an alternative starting methionine for the polypeptides.

Nucleotide sequences of representative OSMRbeta-encoding DNA aredescribed in SEQ ID NO:6 (from nucleotide 368 to 3304), with its deduced979 amino acid sequence described in SEQ ID NO:7. In its entirety, theOSMRbeta polypeptide (SEQ ID NO:7) represents a full-length polypeptidesegment (residue 1 (Met) to residue 979 (Cys) of SEQ ID NO:7. Thedomains and structural features of the OSMRbeta polypeptides are furtherdescribed below.

Analysis of the OSMRbeta polypeptide encoded by the DNA sequence of SEQID NO:6 revealed an open reading frame encoding 979 amino acids (SEQ IDNO:7) comprising a predicted secretory signal peptide of 27 amino acidresidues (residue 1 (Met) to residue 27 (Ala) of SEQ ID NO:7), and amature polypeptide of 952 amino acids (residue 28 (Glu) to residue 979(Cys) of SEQ ID NO:7. In addition to the two WSXWS motifs (SEQ ID NO:3)(corresponding to residues 129 to 133 and residues 415 to 419 of SEQ IDNO:7), the receptor comprises an extracellular domain (residues 28 (Glu)to 739 (Ser) of SEQ ID NO:7); which includes a cytokine-binding domainof approximately 400 amino acid residues (residues 28 (Glu) to 429 (Ala)of SEQ ID NO:7, which includes two linker domains (residues 31 (Pro) to34 (Pro) and residues 343 (Asn) to 347 (Thr)), three regions of cytokinebinding (residues 35 (Val) to 137 (Glu), residues 240 (Pro) to 342(Glu), and residues 348 (Asn) to 429 (Ala), an immuglobulin domain(residues 138 (Val) to 239 (Glu), two penultimate strand regions(residues 106 (His) to 115 (Lys) and residues 398 (Thr) to 405 (Arg) ofSEQ ID NO:7), and a fibronectin type III domain (residues 430 (Pro) to739 (Ser) of SEQ ID NO:7); a transmembrane domain (residues 740 (Met) to761 (Leu) of SEQ ID NO:7); complete intracellular signaling domain(residues 762 (Lys) to 979 (Cys) of SEQ ID NO:7) which contains a “BoxI” signaling site (residues 771 (Tyr) to 777 (Pro) of SEQ ID NO:7), anda “Box II” signaling site (residues 829 (Glu) to 832 (Leu) of SEQ IDNO:7). Those skilled in the art will recognize that these domainboundaries are approximate, and are based on alignments with knownproteins and predictions of protein folding. In addition to thesedomains, conserved receptor features in the encoded receptor include (asshown in SEQ ID NO:7) conserved Trp residues at positions 52 and 353, aconserved Cys residue at position 288, CXW motif (wherein X is any aminoacid) at positions 294-296, and a conserved Arg residue at position 405.The corresponding polynucleotides encoding the OSMRbeta polypeptideregions, domains, motifs, residues and sequences described above are asshown in SEQ ID NO:6.

The presence of transmembrane regions, and conserved and low variancemotifs generally correlates with or defines important structural regionsin proteins. Regions of low variance (e.g., hydrophobic clusters) aregenerally present in regions of structural importance (Sheppard, P. etal., supra.). Such regions of low variance often contain rare orinfrequent amino acids, such as Tryptophan. The regions flanking andbetween such conserved and low variance motifs may be more variable, butare often functionally significant because they may relate to or defineimportant structures and activities such as binding domains, biologicaland enzymatic activity, signal transduction, cell-cell interaction,tissue localization domains and the like.

The regions of conserved amino acid residues in zcytor17, describedabove, can be used as tools to identify new family members. Forinstance, reverse transcription-polymerase chain reaction (RT-PCR) canbe used to amplify sequences encoding the conserved regions from RNAobtained from a variety of tissue sources or cell lines. In particular,highly degenerate primers designed from the zcytor17 sequences areuseful for this purpose. Designing and using such degenerate primers maybe readily performed by one of skill in the art.

The present invention also contemplates a multimeric zcytor17 receptor,as detailed herein, which is capable of intracellular signaling. Suchreceptors may include at least a portion of at least one extracellulardomain of a zcytor17 receptor, and an intracellular domain from azcytor17 receptor or another class I cytokine receptor. In addition tothe extracellular domain of zcytor17, the multimeric cytokine receptorcan also include the extracellular domain of at least a portion of classI cytokine receptor, for instance, the ligand binding domains ofOSMRbeta receptor and/or WSX-1 receptor. Alternatively, the multimericcytokine receptor may include the extracellular domain of anotherreceptor, such as another class I cytokine receptor, and theintracellular domain of zcytor17 to effect intracellular signaling.

The present invention further contemplates a multimeric cytokinereceptor that is soluble. For example, a multimeric cytokine receptormay be, for instance, a heterodimer which includes, for example, aportion of the extracellular domain of zcytor17 and a portion of theextracellular domain of a class I cytokine receptor, such as OSMRbeta(SEQ ID NO:7) and/or WSX-1 (SEQ ID NO:9). Additionally, a solublemultimeric cytokine receptor may also include an affinity tag, such asan immuglobulin F_(c) polypeptide. The soluble multimeric cytokinereceptor can be expressed as a fusion with an immunoglobulin heavy chainconstant region, such as an F_(c) fragment, which contains two constantregion domains and lacks the variable region. Such fusions are typicallysecreted as multimeric molecules wherein the F_(c) portions aredisulfide bonded to each other and two non-Ig polypeptides are arrayedin closed proximity to each other. Fusions of this type can be used forexample, for dimerization, increasing stability and in vivo half-life,to affinity purify ligand, as in vitro assay tool or antagonist.

Through processes of cloning, and proliferation assays described indetail herein, a multimeric cytokine receptor of the present inventionhas been shown to bind a novel ligand polypeptide (zcytor17lig) (SEQ IDNO:2), disclosed in commonly owned U.S. Patent Application Ser. No.60/350,325 and commonly owned U.S. Patent Application Ser. No.60/375,323, with high specificity. Zcytor17lig was isolated from a cDNAlibrary generated from activated human peripheral blood cells (hPBCs),which were selected for CD3. CD3 is a cell surface marker unique tocells of lymphoid origin, particularly T cells.

A zcytor17lig positive clone was isolated, and sequence analysisrevealed that the polynucleotide sequence contained within the plasmidDNA was novel. The secretory signal sequence is comprised of amino acidresidues 1 (Met) to 23 (Ala), and the mature polypeptide is comprised ofamino acid residues 24 (Ser) to 164 (Thr) (as shown in SEQ ID NO:2).Further, N-Terminal sequencing analysis of purified zcytor17lig from293T cells showed an N-terminus at residue 27 (Leu) as shown in SEQ IDNO:2, with the mature polypeptide comprised of amino acid residues 27(Leu) to 164 (Thr) (as shown in SEQ ID NO:2).

In general, cytokines are predicted to have a four-alpha helixstructure, with helices A, C and D being most important inligand-receptor interactions, and are more highly conserved amongmembers of the family. Referring to the human zcytor17lig amino acidsequence shown in SEQ ID NO:2, alignment of human zcytor17lig, humanIL-3, and human cytokine amino acid sequences it is predicted thatzcytor17lig helix A is defined by amino acid residues 38-52; helix B byamino acid residues 83-98; helix C by amino acid residues 104-117; andhelix D by amino acid residues 137-152; as shown in SEQ ID NO:2.Structural analysis suggests that the A/B loop is long, the B/C loop isshort and the C/D loop is long. This loop structure results in anup-up-down-down helical organization. Based on 4-helix bundle structure,the cysteine residues within zcytor17lig that are conserved correspondto amino acid residues 72, 133, and 147 of SEQ ID NO:2; and 74, 137, and151 of SEQ ID NO:11 described herein. Consistent cysteine placement isfurther confirmation of the four-helical-bundle structure. Also highlyconserved in the zcytor17lig is the Glu residue as shown in SEQ ID NO:2at residue 43.

Moreover, the predicted amino acid sequence of murine zcytor17lig shows31% identity to the predicted human protein over the entire length ofthe sequences (SEQ ID NO:2 and SEQ ID NO:11). Based on comparisonbetween sequences of human and murine zcytor17lig conserved residueswere found in the regions predicted to encode alpha helices C and D. Thecorresponding polynucleotides encoding the human zcytor17lig polypeptideregions, domains, motifs, residues and sequences described herein are asshown in SEQ ID NO:1.

While helix D is relatively conserved between human and murinezcytor17lig, helix C is the most conserved. While both species havepredominant acidic amino acids in this region, the differences mayaccount for species specificity in interaction between zcytor17lig andits receptor, zcytor17, comprising monomeric, heterodimeric (e.g.,zcytor17/OSMRbeta, WSX-1/OSMRbeta, zcytor17/WSX-1) or multimeric (e.g.,zcytor17/OSMRbeta/WSX-1) receptors. Loop A/B and helix B of zcytor17ligare marginally conserved, and helix C through Loop C/D into helix D ismost conserved between species; conservation through this regionsuggests that it is functionally significant. The D helices of human andmurine zcytor17lig are also conserved. Zcytor17 receptor antagonists maybe designed through mutations within zcytor17lig helix D. These mayinclude truncation of the protein from residue Thr156 (SEQ ID NO:2), orconservation of residues that confer binding of the ligand to thereceptor, but diminish signaling activity.

Four-helical bundle cytokines are also grouped by the length of theircomponent helices. “Long-helix” form cytokines generally consist ofbetween 24-30 residue helices, and include IL-6, ciliary neutrotrophicfactor (CNTF), leukemia inhibitory factor (LIF) and human growth hormone(hGH). “Short-helix” form cytokines generally consist of between 18-21residue helices and include IL-2, IL-4 and GM-CSF. Zcytor17lig isbelieved to be a new member of the short-helix form cytokine group.Studies using CNTF and IL-6 demonstrated that a CNTF helix can beexchanged for the equivalent helix in IL-6, conferring CTNF-bindingproperties to the chimera. Thus, it appears that functional domains offour-helical cytokines are determined on the basis of structuralhomology, irrespective of sequence identity, and can maintain functionalintegrity in a chimera (Kallen et al., J. Biol. Chem. 274:11859-11867,1999). Therefore, the helical domains of zcytor17lig may be useful forpreparing chimeric fusion molecules, particularly with other short-helixform cytokines to determine and modulate receptor binding specificity.The present invention also envisions fusion proteins engineered withhelix A and/or helix D, and fusion proteins that combine helical andloop domains from other short-form cytokines such as IL-2, IL-4, IL-15,Lif, IL-12, IL-3 and GM-CSF.

The polynucleotide sequence for human IL-2 is shown in SEQ ID NO:176 andthe corresponding amino acid sequence is shown in SEQ ID NO:177. Thesecretory signal sequence is comprised of amino acid residues 1 (Met) to20 (Ser) of SEQ ID NO:177; nucleotides 48 to 107 of SEQ ID NO:176. Themature polypeptide is comprised of amino acid residues 21 (Ala) to 156(Thr) of SEQ ID NO:177; nucleotides 108 to 515 of SEQ ID NO:176. Helix Aof human IL-2 is comprised of amino acid residues 27 (Thr) to 48 (Leu)of SEQ ID NO:177; nucleotides 126 to 191 of SEQ ID NO:176. Helix B ofhuman IL-2 comprises Helix B1 and Helix B2. Helix B1 of human IL-2 iscomprised of amino acid residues 73 (Ala) to 80 (Gln) of SEQ ID NO:177;nucleotides 264 to 287 of SEQ ID NO:176. Helix B2 of human IL-2 iscomprised of amino acid residues 83 (Glu) to 92 (Val) of SEQ ID NO:177;nucleotides 294 to 323 of SEQ ID NO:176. Thus, Helix B (comprisingHelices B1 and B2) of IL-2 is represented by the amino acid sequence ofSEQ ID NO:183 (nucleotide sequence of SEQ ID NO:182) wherein amino acidresidues 9 and 10 can be any amino acid. SEQ ID NO:183 is identical toamino acids 73 (Ala) to 92 (Val) of SEQ ID NO:177 wherein amino acids 81and 82 are any amino acid. In a preferred form, Helix B of IL-2comprises amino acids 73 (Ala) to 92 (Val) of SEQ ID NO:177; nucleotides264 to 323 of SEQ ID NO:176. Helix C of human IL-2 is comprised of aminoacid residues 102 (His) to 116 (Val) of SEQ ID NO:177 nucleotides 351 to395 of SEQ ID NO:176. Helix D of human IL-2 is comprised of amino acidresidues 134 (Thr) to 149 (Gln) of SEQ ID NO:177; nucleotides 447 to 494of SEQ ID NO:176.

The polynucleotide sequence for human IL-4 is shown in SEQ ID NO:178 andthe corresponding amino acid sequence is shown in SEQ ID NO:179. Thesecretory signal sequence is comprised of amino acid residues 1 (Met) to24 (Gly) of SEQ ID NO:179; nucleotides 64 to 135 of SEQ ID NO:178. Themature polypeptide is comprised of amino acid residues 25 (His) to 153(Ser) of SEQ ID NO:179; nucleotides 136 to 522 of SEQ ID NO:178. Helix Aof human IL-4 is comprised of amino acid residues 30 (Thr) to 42 (Thr)of SEQ ID NO:179; nucleotides 151 to 189 of SEQ ID NO:178. Helix B ofhuman IL-4 is comprised of amino acid residues 65 (Glu) to 83 (His) ofSEQ ID NO:179; nucleotides 256 to 312 of SEQ ID NO:178. Helix C of humanIL-4 is comprised of amino acid residues 94 (Ala) to 118 (Ala) of SEQ IDNO:179; nucleotides 343 to 417 of SEQ ID NO:178. Helix D of human IL-4is comprised of amino acid residues 133 (Leu) to 151 (Cys) of SEQ IDNO:179; nucleotides 460 to 516 of SEQ ID NO:178.

The polynucleotide sequence for human GM-CSF is shown in SEQ ID NO:180and the corresponding amino acid sequence is shown in SEQ ID NO:181. Thesecretory signal sequence is comprised of amino acid residues 1 (Met) to17 (Ser) of SEQ ID NO:181; nucleotides 9 to 59 of SEQ ID NO:180. Themature polypeptide is comprised of amino acid residues 18 (Ala) to 144(Glu) of SEQ ID NO:181; nucleotides 60 to 440 of SEQ ID NO:180. Helix Aof human GM-CSF is comprised of amino acid residues 30 (Trp) to 44 (Asn)of SEQ ID NO:181; nucleotides 96 to 140 of SEQ ID NO:180. Helix B ofhuman GM-CSF is comprised of amino acid residues 72 (Leu) to 81 (Gln) ofSEQ ID NO:181; nucleotides 222 to 251 of SEQ ID NO:180. Helix C of humanGM-CSF is comprised of amino acid residues 85 (Gly) to 103 (Gln) of SEQID NO:181; nucleotides 261 to 317 of SEQ ID NO:180. Helix D of humanGM-CSF is comprised of amino acid residues 120 (Phe) to 131 (Leu) of SEQID NO:181; nucleotides 366 to 401 of SEQ ID NO:180.

The amino acid residues comprising helices A, B, C, and D, for humanzcytor17lig, IL-3, IL-2, IL-4, and GM-CSF are shown in Table 1. TABLE 1Helix A Helix B Helix C Helix D zcytor17lig 38-52 83-98 104-117  137-152of SEQ ID NO: 2 IL-3 35-45 73-86 91-103 123-141 of SEQ ID NO: 102 IL-227-48 73-92 102-116  134-149 of SEQ ID NO: 177 or Helix B as describedin SEQ ID NO: 183 IL-4 30-42 65-83 94-118 133-151 of SEQ ID NO: 179GM-CSF 30-44 72-81 85-103 120-131 of SEQ ID NO: 181

The present invention provides polynucleotide molecules, including DNAand RNA molecules that encode the zcytor17 polypeptides disclosed hereinthat can be included in the multimeric cytokine receptor. Those skilledin the art will recognize that, in view of the degeneracy of the geneticcode, considerable sequence variation is possible among thesepolynucleotide molecules. SEQ ID NO:120, SEQ ID NO:121 and SEQ ID NO:122are degenerate DNA sequences that encompass all DNAs that encode thezcytor17 polypeptide of SEQ ID NO:111, SEQ ID NO:109 and SEQ ID NO:5,respectively, and fragments thereof. Those skilled in the art willrecognize that the degenerate sequences of SEQ ID NO:120, SEQ ID NO:121and SEQ ID NO:122 also provide all RNA sequences encoding SEQ ID NO:111,SEQ ID NO:109 and SEQ ID NO:5 by substituting U for T. Thus, zcytor17polypeptide-encoding polynucleotides comprising nucleotide 1 tonucleotide 2196 of SEQ ID NO:120, nucleotide 1 to nucleotide 1947 of SEQID NO:121, and nucleotide 1 to nucleotide 1986 of SEQ ID NO:122 andtheir RNA equivalents are contemplated by the present invention. Table 2sets forth the one-letter codes used within SEQ ID NO:120, SEQ ID NO:121and SEQ ID NO:122 to denote degenerate nucleotide positions.“Resolutions” are the nucleotides denoted by a code letter. “Complement”indicates the code for the complementary nucleotide(s). For example, thecode Y denotes either C or T, and its complement R denotes A or G, Abeing complementary to T, and G being complementary to C. TABLE 2Nucleotide Resolution Complement Resolution A A T T C C G G G G C C T TA A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T WA|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T NA|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:120, SEQ ID NO:121 and SEQ IDNO:122, encompassing all possible codons for a given amino acid, are setforth in Table 3. TABLE 3 One Amino Letter Degenerate Acid Code CodonsCodon Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACCACG ACT ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly GGGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAGGAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGTMGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTCCTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr YTAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln ZSAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequences of SEQ ID NO:111, SEQ ID NO:109 and SEQ ID NO:5; or SEQ IDNO:117 and SEQ ID NO:119. Variant sequences can be readily tested forfunctionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson et al., Gene 13:355-64, 1981; Grosjeanand Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res. 14:3075-87,1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As used herein, the term“preferential codon usage” or “preferential codons” is a term of artreferring to protein translation codons that are most frequently used incells of a certain species, thus favoring one or a few representativesof the possible codons encoding each amino acid (See Table 3). Forexample, the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG,or ACT, but in mammalian cells ACC is the most commonly used codon; inother species, for example, insect cells, yeast, viruses or bacteria,different Thr codons may be preferential. Preferential codons for aparticular species can be introduced into the polynucleotides of thepresent invention by a variety of methods known in the art. Introductionof preferential codon sequences into recombinant DNA can, for example,enhance production of the protein by making protein translation moreefficient within a particular cell type or species. Therefore, thedegenerate codon sequences disclosed in SEQ ID NO:120, SEQ ID NO:121 andSEQ ID NO:122 serve as templates for optimizing expression of zcytor17polynucleotides in various cell types and species commonly used in theart and disclosed herein. Sequences containing preferential codons canbe tested and optimized for expression in various species, and testedfor functionality as disclosed herein.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of zcytor17 RNA. Such tissues and cells areidentified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA77:5201, 1980), and include PBLs, spleen, thymus, bone marrow, prostate,and lymph tissues, human erythroleukemia cell lines, acute monocyticleukemia cell lines, other lymphoid and hematopoietic cell lines, andthe like. Total RNA can be prepared using guanidinium isothiocyanateextraction followed by isolation by centrifugation in a CsCl gradient(Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)⁺ RNA isprepared from total RNA using the method of Aviv and Leder (Proc. Natl.Acad. Sci. USA 69:1408-12, 1972). Complementary DNA (CDNA) is preparedfrom poly(A)⁺ RNA using known methods. In the alternative, genomic DNAcan be isolated. Polynucleotides encoding zcytor17 polypeptides are thenidentified and isolated by, for example, hybridization or polymerasechain reaction (PCR) (Mullis, U.S. Pat. No. 4,683,202).

A full-length clone encoding zcytor17 can be obtained by conventionalcloning procedures. Complementary DNA (cDNA) clones are preferred,although for some applications (e.g., expression in transgenic animals)it may be preferable to use a genomic clone, or to modify a cDNA cloneto include at least one genomic intron. Methods for preparing cDNA andgenomic clones are well known and within the level of ordinary skill inthe art, and include the use of the sequence disclosed herein, or partsthereof, for probing or priming a library. Expression libraries can beprobed with antibodies to zcytor17, receptor fragments, or otherspecific binding partners.

The polynucleotides of the present invention can also be synthesizedusing DNA synthesis machines. Currently the method of choice is thephosphoramidite method. If chemically synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short polynucleotides (60 to 80 bp) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. However, for producinglonger polynucleotides (>300 bp), special strategies are usuallyemployed, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length.

An alternative way to prepare a full-length gene is to synthesize aspecified set of overlapping oligonucleotides (40 to 100 nucleotides).After the 3′ and 5′ short overlapping complementary regions (6 to 10nucleotides) are annealed, large gaps still remain, but the shortbase-paired regions are both long enough and stable enough to hold thestructure together. The gaps are filled and the DNA duplex is completedvia enzymatic DNA synthesis by E. coli DNA polymerase I. After theenzymatic synthesis is completed, the nicks are sealed with T4 DNAligase. Double-stranded constructs are sequentially linked to oneanother to form the entire gene sequence which is verified by DNAsequence analysis. See Glick and Pasternak, Molecular Biotechnology,Principles & Applications of Recombinant DNA, (ASM Press, Washington,D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 andClimie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990. Moreover,other sequences are generally added that contain signals for properinitiation and termination of transcription and translation.

The present invention also provides reagents which will find use indiagnostic applications. For example, the zcytor17lig gene, a probecomprising zcytor17lig DNA or RNA or a subsequence thereof, can be usedto determine if the zcytor17lig gene is present on a human chromosome,such as chromosome 12, or if a gene mutation has occurred. Zcytor17ligis located at the 12q24.31 region of chromosome 12 (Example 13).Detectable chromosomal aberrations at the zcytor17lig gene locusinclude, but are not limited to, aneuploidy, gene copy number changes,loss of heterozygosity (LOH), translocations, insertions, deletions,restriction site changes and rearrangements. Such aberrations can bedetected using polynucleotides of the present invention by employingmolecular genetic techniques, such as restriction fragment lengthpolymorphism (RFLP) analysis, short tandem repeat (STR) analysisemploying PCR techniques, and other genetic linkage analysis techniquesknown in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.;Marian, Chest 108:255-65, 1995).

The precise knowledge of a gene's position can be useful for a number ofpurposes, including: 1) determining if a sequence is part of an existingcontig and obtaining additional surrounding genetic sequences in variousforms, such as YACs, BACs or cDNA clones; 2) providing a possiblecandidate gene for an inheritable disease which shows linkage to thesame chromosomal region; and 3) cross-referencing model organisms, suchas mouse, which may aid in determining what function a particular genemight have.

One of skill in the art would recognize that the 12q24 region isfrequently involved in gross genomic rearrangements, includingtranslocations, deletions, inversions, and duplications, that areassociated with various cancers. The Mitelman Database of ChromosomalAberrations in Cancer, at the Cancer Genome Anatomy Project, NationalInsitutes of Health, Bethesda, Md. located on the Internet lists 199cases of cancers with genomic rearrangements involving 12q24. Of these,most are part of complex karyotypes with other rearrangements; however,in some cases the rearrangement involving 12q24 is the only genomicalteration. Given the expression of the receptor for zcytor17lig oncells of lymphoid and myeloid lineages, it is particularly significantto note that there are at least 4 cases of myeloid leukemia reported inthe literature in which either translocation (2 cases: Yamagata et al,Cancer Genet Cytogenet 97:90-93, 1997; Dunphy and Batanian, Cancer GenetCytogenet 114:51-57, 1999) or duplication (2 cases: Bonomi et al, CancerGenet Cytogenet 108:75-78, 1999) are the sole genomic alteration. Thissuggests that a gene or genes residing within 12q24 could be directlyinvolved in the malignant transformation of these patients' cells.Inappropriate over expression of zcytor17lig could contribute tomalignant transformation by promoting aberrant proliferation ofreceptor-bearing cells, through either autocrine or paracrinemechanisms. Inhibition of zcytor17lig activity could thus inhibit growthof such cells. Alternatively, a genomic rearrangement resulting ininactivation of the zcytor17lig gene may promote malignanttransformation and/or metastasis by removing zcytor17ligimmunoregulatory functions. Indeed, a gene suppressing metastasis inprostate cancer has been mapped to 12q24-qter (Ichikawa et al, Asian JAndrol 2:167-171, 2000). If zcytor17lig is the gene within this regionresponsible for the suppression of metastasis, then zcytor17lig itselfmay have therapeutic value in the treatment of cancer.

A diagnostic could assist physicians in determining the type of diseaseand appropriate associated therapy, or assistance in genetic counseling.As such, the inventive anti-zcytor17lig antibodies, polynucleotides, andpolypeptides can be used for the detection of zcytor17lig polypeptide,mRNA or anti-zcytor17lig antibodies, thus serving as markers and bedirectly used for detecting or genetic diseases or cancers, as describedherein, using methods known in the art and described herein. Further,zcytor17lig polynucleotide probes can be used to detect abnormalities orgenotypes associated with chromosome 12q24.3 deletions andtranslocations associated with human diseases, or other translocationsinvolved with malignant progression of tumors or other 12q24.3mutations, which are expected to be involved in chromosomerearrangements in malignancy; or in other cancers. Similarly,zcytor17lig polynucleotide probes can be used to detect abnormalities orgenotypes associated with chromosome 12 trisomy and chromosome lossassociated with human diseases or spontaneous abortion. Thus,zcytor17lig polynucleotide probes can be used to detect abnormalities orgenotypes associated with these defects.

One of skill in the art would recognize that zcytor17lig polynucleotideprobes are particularly useful for diagnosis of gross chromosomalabnormalities associated with loss of heterogeneity (LOH), chromosomegain (e.g., trisomy), translocation, DNA amplification, and the like.Translocations within chromosomal locus 12q24.3 wherein the zcytor17liggene is located are known to be associated with human disease. Forexample, 12q24 deletions and translocations, duplications and trisomyare associated with cancers as discussed above. Thus, since thezcytor17lig gene maps to this critical region, zcytor17ligpolynucleotide probes of the present invention can be used to detectabnormalities or genotypes associated with 12q24 translocation, deletionand trisomy, and the like, described above.

As discussed above, defects in the zcytor17lig gene itself may result ina heritable human disease state. Molecules of the present invention,such as the polypeptides, antagonists, agonists, polynucleotides andantibodies of the present invention would aid in the detection,diagnosis prevention, and treatment associated with a zcytor17liggenetic defect. In addition, zcytor17lig polynucleotide probes can beused to detect allelic differences between diseased or non-diseasedindividuals at the zcytor17lig chromosomal locus. As such, thezcytor17lig sequences can be used as diagnostics in forensic DNAprofiling.

In general, the diagnostic methods used in genetic linkage analysis, todetect a genetic abnormality or aberration in a patient, are known inthe art. Analytical probes will be generally at least 20 nt in length,although somewhat shorter probes can be used (e.g., 14-17 nt). PCRprimers are at least 5 nt in length, preferably 15 or more, morepreferably 20-30 nt. For gross analysis of genes, or chromosomal DNA, azcytor17lig polynucleotide probe may comprise an entire exon or more.Exons are readily determined by one of skill in the art by comparingzcytor17lig sequences (SEQ ID NO:1) with the genomic DNA for mousezcytor17lig (SEQ ID NO:76). In general, the diagnostic methods used ingenetic linkage analysis, to detect a genetic abnormality or aberrationin a patient, are known in the art. Most diagnostic methods comprise thesteps of (a) obtaining a genetic sample from a potentially diseasedpatient, diseased patient or potential non-diseased carrier of arecessive disease allele; (b) producing a first reaction product byincubating the genetic sample with a zcytor17lig polynucleotide probewherein the polynucleotide will hybridize to complementarypolynucleotide sequence, such as in RFLP analysis or by incubating thegenetic sample with sense and antisense primers in a PCR reaction underappropriate PCR reaction conditions; (iii) visualizing the firstreaction product by gel electrophoresis and/or other known methods suchas visualizing the first reaction product with a zcytor17ligpolynucleotide probe wherein the polynucleotide will hybridize to thecomplementary polynucleotide sequence of the first reaction; and (iv)comparing the visualized first reaction product to a second controlreaction product of a genetic sample from wild type patient, or a normalor control individual. A difference between the first reaction productand the control reaction product is indicative of a genetic abnormalityin the diseased or potentially diseased patient, or the presence of aheterozygous recessive carrier phenotype for a non-diseased patient, orthe presence of a genetic defect in a tumor from a diseased patient, orthe presence of a genetic abnormality in a fetus or pre-implantationembryo. For example, a difference in restriction fragment pattern,length of PCR products, length of repetitive sequences at thezcytor17lig genetic locus, and the like, are indicative of a geneticabnormality, genetic aberration, or allelic difference in comparison tothe normal wild type control. Controls can be from unaffected familymembers, or unrelated individuals, depending on the test andavailability of samples. Genetic samples for use within the presentinvention include genomic DNA, mRNA, and cDNA isolated from any tissueor other biological sample from a patient, which includes, but is notlimited to, blood, saliva, semen, embryonic cells, amniotic fluid, andthe like. The polynucleotide probe or primer can be RNA or DNA, and willcomprise a portion of SEQ ID NO:1, the complement of SEQ ID NO:1, or anRNA equivalent thereof. Such methods of showing genetic linkage analysisto human disease phenotypes are well known in the art. For reference toPCR based methods in diagnostics see generally, Mathew (ed.), Protocolsin Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCRProtocols: Current Methods and Applications (Humana Press, Inc. 1993),Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc. 1996),Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc.1998), Lo (ed.), Clinical Applications of PCR (Humana Press, Inc. 1998),and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998).

Mutations associated with the zcytor17lig locus can be detected usingnucleic acid molecules of the present invention by employing standardmethods for direct mutation analysis, such as restriction fragmentlength polymorphism analysis, short tandem repeat analysis employing PCRtechniques, amplification-refractory mutation system analysis,single-strand conformation polymorphism detection, RNase cleavagemethods, denaturing gradient gel electrophoresis, fluorescence-assistedmismatch analysis, and other genetic analysis techniques known in theart (see, for example, Mathew (ed.), Protocols in Human MolecularGenetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995),Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996),Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc.1996), Landegren (ed.), Laboratory Protocols for Mutation Detection(Oxford University Press 1996), Birren et al. (eds.), Genome Analysis,Vol 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),Dracopoli et at. (eds.), Current Protocols in Human Genetics (John Wiley& Sons 1998), and Richards and Ward, “Molecular Diagnostic Testing,” inPrinciples of Molecular Medicine, pages 83-88 (Humana Press, Inc. 1998).Direct analysis of an zcytor17lig gene for a mutation can be performedusing a subject's genomic DNA. Methods for amplifying genomic DNA,obtained for example from peripheral blood lymphocytes, are well-knownto those of skill in the art (see, for example, Dracopoli et at. (eds.),Current Protocols in Human Genetics, at pages 7.1.6 to 7.1.7 (John Wiley& Sons 1998)).

The present invention further provides counterpart polypeptides andpolynucleotides from other species (orthologs). These species include,but are not limited to mammalian, avian, amphibian, reptile, fish,insect and other vertebrate and invertebrate species. Of particularinterest are zcytor17 polypeptides from other mammalian species,including murine, porcine, ovine, bovine, canine, feline, equine, andother primate polypeptides. Orthologs of human zcytor17 can be clonedusing information and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses zcytor17 as disclosed herein. Suitable sources of mRNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue or cell line. A zcytor17-encoding cDNA can then beisolated by a variety of methods, such as by probing with a complete orpartial human cDNA or with one or more sets of degenerate probes basedon the disclosed sequences. A cDNA can also be cloned using PCR (Mullis,supra.), using primers designed from the representative human zcytor17sequence disclosed herein. Within an additional method, the cDNA librarycan be used to transform or transfect host cells, and expression of thecDNA of interest can be detected with an antibody to zcytor17polypeptide. Similar techniques can also be applied to the isolation ofgenomic clones.

A polynucleotide sequence for the mouse ortholog of human zcytor17 hasbeen identified and is shown in SEQ ID NO:116 and the correspondingamino acid sequence shown in SEQ ID NO:117. Analysis of the mousezcytor17 polypeptide encoded by the DNA sequence of SEQ ID NO:116revealed an open reading frame encoding 662 amino acids (SEQ ID NO:117)comprising a predicted secretory signal peptide of 45 amino acidresidues (residue 1 (Met) to residue 45 (Ala) of SEQ ID NO:117), and amature polypeptide of 617 amino acids (residue46 (Val) to residue 662(Cys) of SEQ ID NO:117). Moreover, an additional Met residue, Met (28)can be used as a starting methionine; comprising a second predictedsecretory signal peptide of 18 amino acid residues (residue 28 (Met) toresidue 45 (Ala) of SEQ ID NO:117), and the same mature polypeptide of617 amino acids (residue46 (Val) to residue 662 (Cys) of SEQ ID NO:117.In addition to the WSXWS motif (SEQ ID NO:3) corresponding to residues224-228 of SEQ ID NO:117, the receptor comprises an extracellular domainfrom residues 46 (Val) to 533 (Glu) of SEQ ID NO:117) that includes acytokine-binding domain of approximately 200 amino acid residues(residues 46 (Val) to 240 (Pro) of SEQ ID NO:117) and a fibronectin IIIdomain (residues 241 (His) to 533 (Glu) of SEQ ID NO:117); a CXW motif(residues 66 (Cys) to 68 (Trp) of SEQ ID NO:117); a domain linker(residues 142 (Thr) to 145 (Pro) of SEQ ID NO:117); a penultimate strandregion (residues 207 (Phe) to 215 (Arg) of SEQ ID NO:117); atransmembrane domain (residues 534 (Ile) to 550 (Ile) of SEQ ID NO:117);complete intracellular signaling domain (residues 551 (Lys) to 662 (Cys)of SEQ ID NO:117) which contains a “Box I” signaling site (residues 568(Cys) to 574 (Pro) of SEQ ID NO:117), and a “Box II” signaling site(residues 628 (Glu) to 631 (leu) of SEQ ID NO:117). Conserved residuescommon to class I cytokine receptors, are at residues 56 (Cys), 187(Trp), and 215 (Arg). A comparison of the human and mouse amino acidsequences reveals that both the human and orthologous polypeptidescontain corresponding structural features described above (and, see,FIG. 2). The mature sequence for the mouse zcytor17 begins at Val₄₆ (asshown in SEQ ID NO:117), which corresponds to Ala₃₃ (as shown in SEQ IDNO:5) in the human sequence. There is about 61% identity between themouse and human sequences over the entire amino acid sequencecorresponding to SEQ ID NO:5 and SEQ ID NO:117. The above percentidentity was determined using a FASTA program with ktup=1, gap openingpenalty=12, gap extension penalty=2, and substitution matrix=BLOSUM62,with other parameters set as default. The corresponding polynucleotidesencoding the mouse zcytor17 polypeptide regions, domains, motifs,residues and sequences described above are as shown in SEQ ID NO:116.

Moreover, a truncated soluble form of the mouse zcytor17 receptorpolypeptide appears to be naturally expressed. A polynucleotide sequencefor a truncated soluble form of the mouse zcytor17 receptor has beenidentified and is shown in SEQ ID NO:118 and the corresponding aminoacid sequence shown in SEQ ID NO:119. Analysis of the truncated solublemouse zcytor17 polypeptide encoded by the DNA sequence of SEQ ID NO:118revealed an open reading frame encoding 547 amino acids (SEQ ID NO:119)comprising a predicted secretory signal peptide of 45 amino acidresidues (residue 1 (Met) to residue 45 (Ala) of SEQ ID NO:119), and amature polypeptide of 502 amino acids (residue46 (Val) to residue 547(Val) of SEQ ID NO:119). Moreover, an additional Met residue, Met (28)can be used as a starting methionine; comprising a second predictedsecretory signal peptide of 18 amino acid residues (residue 28 (Met) toresidue 45 (Ala) of SEQ ID NO:119), and the same mature polypeptide of502 amino acids (residue46 (Val) to residue 547 (Val) of SEQ ID NO:119.In addition to the WSXWS motif (SEQ ID NO:3) corresponding to residues224-228 of SEQ ID NO:119, the receptor comprises an extracellular domainfrom residues 46 (Val) to 533 (Trp) of SEQ ID NO:119) that includes acytokine-binding domain of approximately 200 amino acid residues(residues 46 (Val) to 240 (Pro) of SEQ ID NO:119) and a fibronectin IIIdomain (residues 241 (His) to 533 (Trp) of SEQ ID NO:119); a CXW motif(residues 66 (Cys) to 68 (Trp) of SEQ ID NO:119); a domain linker(residues 142 (Thr) to 145 (Pro) of SEQ ID NO:119); a penultimate strandregion (residues 207 (Phe) to 215 (Arg) of SEQ ID NO:119); and aC-terminal tail region (residues 534 (Leu) to 547 (Val). Conservedresidues common to class I cytokine receptors, are at residues 56 (Cys),187 (Trp), and 215 (Arg). A comparison of the human and mouse amino acidsequences, including the truncated soluble mouse zcytor17, reveals thatboth the human and orthologous polypeptides contain correspondingstructural features described above (and, see, FIG. 2). Thecorresponding polynucleotides encoding the truncated soluble mousezcytor17 polypeptide regions, domains, motifs, residues and sequencesdescribed above are as shown in SEQ ID NO:118.

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NO:110, SEQ ID NO:108 and SEQ ID NO:4 represent alleles of humanzcytor17 and that allelic variation and alternative splicing areexpected to occur. Allelic variants of this sequence can be cloned byprobing cDNA or genomic libraries from different individuals accordingto standard procedures. Allelic variants of the DNA sequence shown inSEQ ID NO:110, SEQ ID NO:108 or SEQ ID NO:4, including those containingsilent mutations and those in which mutations result in amino acidsequence changes, are within the scope of the present invention, as areproteins which are allelic variants of SEQ ID NO:111, SEQ ID NO:109, SEQID NO:5 SEQ ID NO:117 or SEQ ID NO:119. cDNAs generated fromalternatively spliced mRNAs, which retain the properties of the zcytor17polypeptide are included within the scope of the present invention, asare polypeptides encoded by such cDNAs and mRNAs. Allelic variants andsplice variants of these sequences can be cloned by probing cDNA orgenomic libraries from different individuals or tissues according tostandard procedures known in the art. For example, the short-form andlong-form soluble zcytor17 receptors described above, and in SEQ IDNO:112 and SEQ ID NO:113 or SEQ ID NO:114 and SEQ ID NO:115 can beconsidered allelic or splice variants of zcytor17.

The present invention also provides isolated zcytor17 polypeptides thatare substantially similar to the polypeptides of SEQ ID NO:111, SEQ IDNO:109 or SEQ ID NO:5 and their orthologs, e.g., SEQ ID NO:117 and SEQID NO:119. The term “substantially similar” is used herein to denotepolypeptides having at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or greater than 99% sequence identity to the sequences shown in SEQID NO:111, SEQ ID NO:109 or SEQ ID NO:5 or their orthologs, e.g., SEQ IDNO:117 and SEQ ID NO:119. Such polypeptides will more preferably be atleast 90% identical, and most preferably 95% or more identical to SEQ IDNO:111, SEQ ID NO:109 and SEQ ID NO:5 or its orthologs.) Percentsequence identity is determined by conventional methods. See, forexample, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 4 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as:$\frac{{Total}\quad{number}\quad{of}\quad{identical}\quad{matches}}{\begin{bmatrix}{{length}\quad{of}\quad{the}\quad{longer}\quad{sequence}\quad{plus}\quad{the}} \\{{number}\quad{of}\quad{gaps}\quad{introduced}\quad{into}\quad{the}\quad{longer}} \\{{sequence}\quad{in}\quad{order}\quad{to}\quad{align}\quad{the}\quad{two}\quad{sequences}}\end{bmatrix}} \times 100$ TABLE 4 A R N D C Q E G H I L K M F P S T W YV A 4 R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 00 2 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3−1 −3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2−1 −3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3−3 −1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 10 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1−2 −1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2−2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3−3 3 1 −2 1 −1 −2 −2 0 −3 −1 4

Sequence identity of polynucleotide molecules is determined by similarmethods using a ratio as disclosed above.

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant zcytor17. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:111, SEQ IDNO:109, SEQ ID NO:5, SEQ ID NO:117 and SEQ ID NO:119) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62, with other parameters set as default.These parameters can be introduced into a FASTA program by modifying thescoring matrix file (“SMATRIX”), as explained in Appendix 2 of Pearson,Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other FASTA programparameters set as default.

The BLOSUM62 table (Table 4) is an amino acid substitution matrixderived from about 2,000 local multiple alignments of protein sequencesegments, representing highly conserved regions of more than 500 groupsof related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies canbe used to define conservative amino acid substitutions that may beintroduced into the amino acid sequences of the present invention.Although it is possible to design amino acid substitutions based solelyupon chemical properties (as discussed below), the language“conservative amino acid substitution” preferably refers to asubstitution represented by a BLOSUM62 value of greater than −1. Forexample, an amino acid substitution is conservative if the substitutionis characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to thissystem, preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), whilemore preferred conservative amino acid substitutions are characterizedby a BLOSUM62 value of at least 2 (e.g., 2 or 3).

Variant zcytor17 polypeptides or substantially homologous zcytor17polypeptides are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table5) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. The present inventionthus includes polypeptides that comprise a sequence that is at least80%, preferably at least 90%, and more preferably 95% or more identicalto the corresponding region of SEQ ID NO:111, SEQ ID NO:109, SEQ IDNO:5, SEQ ID NO:117 or SEQ ID NO:119 excluding the tags, extension,linker sequences and the like. Polypeptides comprising affinity tags canfurther comprise a proteolytic cleavage site between the zcytor17polypeptide and the affinity tag. Suitable sites include thrombincleavage sites and factor Xa cleavage sites. TABLE 5 Conservative aminoacid substitutions Basic: arginine lysine histidine Acidic: glutamicacid aspartic acid Polar: glutamine asparagine Hydrophobic: leucineisoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small:glycine alanine serine threonine methionine

The present invention provides an isolated an isolated multimericcytokine receptor comprising a polypeptide having at least 90 percentsequence identity with SEQ ID NO:111, SEQ ID NO:109, or SEQ ID NO:5; andat least a portion of at least one class I cytokine receptor, whereinthe multimeric cytokine receptor binds to at least a portion of SEQ IDNO:2. The polypeptide having at least 90 percent sequence identity withSEQ ID NO:111 may include, for instance, amino acid residue 20 to aminoacid residue 227 of SEQ ID NO:111, amino acid residue 20 to amino acidresidue 227 of SEQ ID NO:519, amino acid residue 20 to amino acidresidue 543 of SEQ ID NO:111, amino acid residue 544 to amino acidresidue 732 of SEQ ID NO:111, amino acid residue 544 to amino acidresidue 649 of SEQ ID NO:109, amino acid residue 20 to amino acidresidue 732 of SEQ ID NO:111, amino acid residue 20 to amino acidresidue 649 of SEQ ID NO:109, and combinations thereof. The at least aportion of at least one class I cytokine receptor includes, for example,OSMRbeta (SEQ ID NO:7) and/or WSX-1 (SEQ ID NO:9). For example, the atleast a portion of at least one class I cytokine receptor comprising atleast a portion of SEQ ID NO:7 may comprise amino acid residue 28 toamino acid residue 429 of SEQ ID NO:7, amino acid residue 35 to aminoacid residue 137 of SEQ ID NO:7, amino acid residue 240 to amino acidresidue 342 of SEQ ID NO:7, amino acid residue 348 to amino acid residue429 of SEQ ID NO:7, amino acid residue 28 to amino acid residue 739 ofSEQ ID NO:7, amino acid residue 28 to amino acid residue 761 of SEQ IDNO:7, amino acid residue 762 to amino acid residue 979 of SEQ ID NO:7,and/or combinations thereof. The multimeric cytokine receptor may be aheterodimer, trimer, tetramer, pentamer, or the like. In addition, themultimeric cytokine receptor may be soluble, immobilized on a solidsupport, or membrane-bound. Optionally, the multimeric cytokine receptormay antagonize an activity of SEQ ID NO:2, such as inhibit or reduce theproliferation of hematopoietic, immune, and/or inflammatory cells, orinhibit or reduce the enhancement of the hematopoietic, immune, and/orinflammatory process, or inhibit or reduce the differentiation ofhematopoietic cells, for instance lymphoid cells such as monocyticcells, macrophages and/or T cells. The multimeric cytokine receptor ofthe present invention may also comprise an affinity tag selected fromthe group of polyhistidine, protein A, glutathione S transferase,Glu-Glu, substance P, Flag™ peptide, streptavidin binding peptide, andan immunoglobulin F_(c) polypeptide.

The present invention also provides for a ligand/receptor complexcomprising a polypeptide which includes at least a portion of SEQ IDNO:2; and a soluble multimeric cytokine receptor comprising at least aportion of at least one polypeptide selected from the group of SEQ IDNO:111, SEQ ID NO:109, SEQ ID NO:7, and SEQ ID NO:9, wherein thepolypeptide is attached to the soluble multimeric cytokine receptor. Thesoluble multimeric cytokine receptor may comprise the extracellulardomain and/or transmembrane domain of zcytor17 (SEQ ID NO:111), OSMRbeta(SEQ ID NO:7), and/or WSX-1 (SEQ ID NO:9). For example, the solublemultimeric cytokine receptor may comprise amino acid residue 20 to 227to SEQ ID NO:111, amino acid residue 20 to 519 to SEQ ID NO:111, aminoacid residue 20 to 543 to SEQ ID NO:111, amino acid residue 28 to 739 toSEQ ID NO:7, amino acid residue 28 to 429 to SEQ ID NO:7, amino acidresidue 35 to 137 to SEQ ID NO:7, amino acid residue 240 to 342 to SEQID NO:7, amino acid residue 348 to 429 to SEQ ID NO:7, or combinationsthereof. The soluble multimeric cytokine receptor may be a heterodimer,trimer, tetramer, pentamer, or the like. The multimeric cytokinereceptor of the present invention may also comprise an affinity tag asdescribed herein. The polypeptide of the ligand/receptor complex maycomprise amino acid residues of SEQ ID NO:2 selected from the group of38 to 152, 27 to 164, 24 to 164, 1 to 164, 38 to 52, 83 to 98, 104 to117, 137 to 152, and combinations thereof.

The ligand/receptor complex of the present may also comprise a fusionprotein. The fusion protein may comprise at least four polypeptides,wherein the order of polypeptides from N-terminus to C-terminus are afirst polypeptide comprising amino acid residues 38-52 of SEQ ID NO:2; afirst spacer of 6-27 amino acid residues; a second polypeptidecomprising amino acid residues selected from the group of (a) IL-2 helixB residues of SEQ ID NO:183; (b) IL-4 helix B residues 65-83 of SEQ IDNO:179; (c) IL-3 helix B residues 73-86 of SEQ ID NO:102; (d) GM-CSFhelix B residues 72-81 of SEQ ID NO:181; and (e) amino acid residues83-98 of SEQ ID NO:2; a second spacer of 5-11 amino acid residues; athird polypeptide comprising amino acid residues selected from the groupof (a) IL-2 helix C residues 102-116 of SEQ ID NO:177; (b) IL-4 helix Cresidues 94-118 of SEQ ID NO:179; (c) IL-3 helix C residues 91-103 ofSEQ ID NO:102; (d) GM-CSF helix C residues 85-103 of SEQ ID NO:181; and(e) amino acid residues 104-117 of SEQ ID NO:2; a third spacer of 3-29amino acid residues; and a fourth polypeptide comprising amino acidresidues selected from the group of (a) IL-2 helix D residues 134-149 ofSEQ ID NO:177 ; (b) IL-3 helix D residues 123-141 of SEQ ID NO:102; (c)IL-4 helix D residues 133-151 of SEQ ID NO:179; (d) GM-CSF helix Dresidues 120-131 of SEQ ID NO:181; and (e) amino acid residues 137-152of SEQ ID NO:2; and a multimeric cytokine receptor comprising at least aportion of at least one polypeptide selected from the group of SEQ IDNO:111, SEQ ID NO:109, SEQ ID NO:7, and SEQ ID NO:9; wherein the fusionprotein is attached to the multimeric cytokine receptor.

Alternatively, the fusion protein may comprise at least fourpolypeptides, wherein the order of polypeptides from N-terminus toC-terminus are a first polypeptide comprising amino acid residuesselected from a group of (a) IL-2 helix A residues 27-48 of SEQ IDNO:177; (b) IL-4 helix A residues 30-42 of SEQ ID NO:179; (c) IL-3 helixA residues 35-45 of SEQ ID NO:102; (d) GM-CSF helix A residues 30-44 ofSEQ ID NO:181; and (e) amino acids residues 38-52 of SEQ ID NO:2; afirst spacer of 6-27 amino acid residues; a second polypeptidecomprising amino acid residues selected from the group of (a) IL-2 helixB residues of SEQ ID NO:183; (b); IL-4 helix B residues 65-83 of SEQ IDNO:179; (c) IL-3 helix B residues 73-86 of SEQ ID NO:102; (d) GM-CSFhelix B residues 72-81 of SEQ ID NO:181; and (e) amino acid residues83-98 of SEQ ID NO:2; a second spacer of 5-11 amino acid residues; athird polypeptide comprising amino acid residues selected from the groupof (a) IL-2 helix C residues 102-116 of SEQ ID NO:177; (b) IL-4 helix Cresidues 94-118 of SEQ ID NO:179; (c) IL-3 helix C residues 91-103 ofSEQ ID NO:102; (d) GM-CSF helix C residues 85-103 of SEQ ID NO:181; and(e) amino acid residues 104-117 of SEQ ID NO:2; a third spacer of 3-29amino acid residues; and a fourth polypeptide comprising amino acidresidues from 137-152 of SEQ ID NO:2; and a multimeric cytokine receptorcomprising at least a portion of at least one polypeptide selected fromthe group of SEQ ID NO:111, SEQ ID NO:109, SEQ ID NO:7, and SEQ ID NO:9;wherein the fusion protein is attached to the multimeric cytokinereceptor. A multimeric cytokine receptor may comprise at least one ofthe following polypeptides of SEQ ID NO:111, SEQ ID NO:109, SEQ ID NO:7,SEQ ID NO:9, or the extracellular domains thereof. The ligand/receptorcomplex can be soluble and may additionally include an affinity tag asdescribed herein.

The present invention also provides an isolated and purifiedpolynucleotide that encodes a polypeptide comprising an amino acidsequence having at least 90 percent sequence identity with SEQ IDNO:111, SEQ ID NO:109, or SEQ ID NO:5, wherein the polypeptide and atleast a portion of at least one class I cytokine receptor form amultimeric cytokine receptor, and wherein the multimeric cytokinereceptor binds to at least a portion of SEQ ID NO:2. The presentinvention also provides an isolated and purified polynucleotide thatencodes a polypeptide comprising at least a portion of at least one ofSEQ ID NO:111, SEQ ID NO:109, or SEQ ID NO:5, wherein the polypeptideand at least a portion of a class I cytokine receptor form a multimericcytokine receptor, and wherein the multimeric cytokine receptor binds toat least a portion of SEQ ID NO:2. The polynucleotide may encode apolypeptide that is included in a soluble multimeric cytokine receptorand that may also include an affinity tag as described herein. Thepolypeptide may comprise, for example, amino acid residue 20 to 227 toSEQ ID NO:111, amino acid residue 20 to 519 to SEQ ID NO:111, amino acidresidue 20 to 543 to SEQ ID NO:111, and/or combinations thereof. Inaddition, the at least a portion of at least one class I cytokinereceptor may comprise, for example, amino acid residue 28 to 739 to SEQID NO:7, amino acid residue 28 to 429 to SEQ ID NO:7, amino acid residue35 to 137 to SEQ ID NO:7, amino acid residue 240 to 342 to SEQ ID NO:7,amino acid residue 348 to 429 to SEQ ID NO:7, and/or combinationsthereof. The soluble multimeric cytokine receptor may be a heterodimer,trimer, tetramer, pentamer, or the like. The at least a portion of SEQID NO:2 may include, for instance amino acid residues of SEQ ID NO:2selected from the group of 38 to 152, 27 to 164, 24 to 164, 1 to 164, 38to 52, 83 to 98, 104 to 117, 137 to 152, and combinations thereof.Optionally, the multimeric cytokine receptor may antagonize an activityof SEQ ID NO:2 as described herein.

The present invention further provides a variety of other polypeptidefusions and related multimeric proteins comprising one or morepolypeptide fusions. For example, a zcytor17 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Preferred dimerizing proteins in this regardinclude immunoglobulin constant region domains. Immunoglobulin-zcytor17polypeptide fusions can be expressed in genetically engineered cells toproduce a variety of multimeric zcytor17 analogs. Auxiliary domains canbe fused to zcytor17 polypeptides to target them to specific cells,tissues, or macromolecules (e.g., collagen). A zcytor17 polypeptide canbe fused to two or more moieties, such as an affinity tag forpurification and a targeting domain. Polypeptide fusions can alsocomprise one or more cleavage sites, particularly between domains. See,Tuan et al., Connective Tissue Research 34:1-9, 1996. For example, oneor more domains from zcytor17 soluble receptor can be joined to asoluble cytokine receptor, such as OSMRbeta and/or WSX-1, which mayenhance their biological properties or efficiency of production.Additionally, the soluble multimeric cytokine receptor may furtherinclude an affinity tag. An affinity tag can be, for example, a tagselected from the group of polyhistidine, protein A, glutathione Stransferase, Glu-Glu, substance P, Flag™ peptide, streptavidin bindingpeptide, and an immunoglobulin F_(c) polypeptide.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is carried outin a cell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Methods Enzymol 202:301, 1991; Chung etal., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci.USA 90:10145-9, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-8, 1996). Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for zcytor17 amino acidresidues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad.Sci. USA 88:4498-502, 1991). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (e.g.ligand binding and signal transduction) as disclosed below to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. Sites ofligand-receptor, protein-protein or other biological interaction canalso be determined by physical analysis of structure, as determined bysuch techniques as nuclear magnetic resonance, crystallography, electrondiffraction or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904,1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities ofessential amino acids can also be inferred from analysis of homologieswith related receptors.

Determination of amino acid residues that are within regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to, alignment of multiple sequences withhigh amino acid or nucleotide identity and computer analysis usingavailable software (e.g., the Insight II® viewer and homology modelingtools; MSI, San Diego, Calif.), secondary structure propensities, binarypatterns, complementary packing and buried polar interactions (Barton,Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., CurrentOpin. Struct. Biol. 6:3-10, 1996). In general, when designingmodifications to molecules or identifying specific fragmentsdetermination of structure will be accompanied by evaluating activity ofmodified molecules.

Amino acid sequence changes are made in zcytor17 polypeptides so as tominimize disruption of higher order structure essential to biologicalactivity. For example, when the zcytor17 polypeptide comprises one ormore helices, changes in amino acid residues will be made so as not todisrupt the helix geometry and other components of the molecule wherechanges in conformation abate some critical function, for example,binding of the molecule to its binding partners. The effects of aminoacid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same disulfide bonding patternas the standard molecule folding would be affected. Another well knownand accepted method for measuring folding is circular dichrosism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructural similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

A Hopp/Woods hydrophilicity profile of the zcytor17 protein sequence asshown in SEQ ID NO:2, SEQ ID NO:46, SEQ ID NO:54, SEQ ID NO:57 and SEQID NO:93 can be generated (Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier etal., Protein Engineering 11:153-169, 1998). See, FIG. 1. The profile isbased on a sliding six-residue window. Buried G, S, and T residues andexposed H, Y, and W residues were ignored. For example, in zcytor17,hydrophilic regions include amino acid residues 43 through 48 of SEQ IDNO:2 and SEQ ID NO:46 (residues 56 through 61 of SEQ ID NO:54), aminoacid residues 157 through 162 of SEQ ID NO:2 and SEQ ID NO:46 (residues170 through 175 of SEQ ID NO:54), amino acid residues 158 through 163 ofSEQ ID NO:2 and SEQ ID NO:46 (residues 171 through 176 of SEQ ID NO:54),amino acid residues 221 through 226 of SEQ ID NO:2 and SEQ ID NO:46(residues 234 through 239 of SEQ ID NO:54), and amino acid residues 426through 431 of SEQ ID NO:2 and SEQ ID NO:46 (residues 439 through 444 ofSEQ ID NO:54).

Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a zcytor17 polypeptide, so as not todisrupt the overall structural and biological profile. Of particularinterest for replacement are hydrophobic residues selected from thegroup consisting of Val, Leu and Ile or the group consisting of Met,Gly, Ser, Ala, Tyr and Trp. For example, residues tolerant ofsubstitution could include such residues as shown in SEQ ID NO:2, SEQ IDNO:46, SEQ ID NO:54, SEQ ID NO:57 and SEQ ID NO:93. However, Cysteineresidues would be relatively intolerant of substitution.

The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between class I cytokine receptor familymembers with zcytor17. Using methods such as “FASTA” analysis describedpreviously, regions of high similarity are identified within a family ofproteins and used to analyze amino acid sequence for conserved regions.An alternative approach to identifying a variant zcytor17 polynucleotideon the basis of structure is to determine whether a nucleic acidmolecule encoding a potential variant zcytor17 polynucleotide canhybridize to a nucleic acid molecule having the nucleotide sequence ofSEQ ID NO:1, SEQ ID NO:45 or SEQ ID NO:53, as discussed above.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity as disclosed below to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., J.Biol. Chem. 271:4699 (1996).

The present invention also includes a multimeric cytokine receptor whichincludes functional fragments of zcytor17 polypeptides and nucleic acidmolecules encoding such functional fragments. A “functional” zcytor17 orfragment thereof defined herein is characterized by its ability tomediate proliferative or differentiating activity, by its ability toinduce or inhibit specialized cell functions, or by its ability to bindspecifically to an anti-zcytor17 antibody or zcytor17 ligand (eithersoluble or immobilized). Moreover, functional fragments also include thesignal peptide, intracellular signaling domain, and the like. Aspreviously described herein, zcytor17 is characterized by a class Icytokine receptor structure. Thus, the present invention furtherprovides fusion proteins encompassing: (a) polypeptide moleculescomprising an extracellular domain, cytokine-binding domain, orintracellular domain described herein; and (b) functional fragmentscomprising one or more of these domains. The other polypeptide portionof the fusion protein may comprise at least a portion of one or more ofanother class I cytokine receptor, for example, gp130, LIF, IL-12,WSX-1, IL-2 receptor β-subunit and the β-common receptor (i.e., IL3,IL-5, and GM-CSF receptor β-subunits), or by a non-native and/or anunrelated secretory signal peptide that facilitates secretion of thefusion protein.

Routine deletion analyses of nucleic acid molecules can be performed toobtain functional fragments of a nucleic acid molecule that encodes azcytor17 polypeptide. As an illustration, DNA molecules having thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:45 or SEQ ID NO:53 orfragments thereof, can be digested with Bal31 nuclease to obtain aseries of nested deletions. These DNA fragments are then inserted intoexpression vectors in proper reading frame, and the expressedpolypeptides are isolated and tested for zcytor17 activity, or for theability to bind anti-zcytor17 antibodies or zcytor17 ligand. Onealternative to exonuclease digestion is to use oligonucleotide-directedmutagenesis to introduce deletions or stop codons to specify productionof a desired zcytor17 fragment. Alternatively, particular fragments of azcytor17 polynucleotide can be synthesized using the polymerase chainreaction.

Standard methods for identifying functional domains are well-known tothose of skill in the art. For example, studies on the truncation ateither or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993);Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (ijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation 1, Boynton et al.,(eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol.Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol 50:1295 (1995); and Meiselet al., Plant Molec. Biol. 30:1 (1996).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/062045) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants of the disclosed zcytor17 DNA and polypeptide sequences can begenerated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized zcytor17 receptor polypeptides in host cells.Preferred assays in this regard include cell proliferation assays andbiosensor-based ligand-binding assays, which are described below.Mutagenized DNA molecules that encode active receptors or portionsthereof (e.g., ligand-binding fragments, signaling domains, and thelike) can be recovered from the host cells and rapidly sequenced usingmodern equipment. These methods allow the rapid determination of theimportance of individual amino acid residues in a polypeptide ofinterest, and can be applied to polypeptides of unknown structure.

The present invention also provides a novel multimeric cytokine receptorin which a segment comprising at least a portion of one or more of thedomains of zcytor17, for instance, secretory, extracellular,transmembrane, and intracellular, is fused to another polypeptide, forexample, an extracellular domain of a class I cytokine receptor, such asOSMRbeta and/or WSX-1. Fusion is preferably done by splicing at the DNAlevel to allow expression of chimeric molecules in recombinantproduction systems. The resultant molecules are then assayed for suchproperties as improved solubility, improved stability, prolongedclearance half-life, improved expression and secretion levels, andpharmacodynamics. Such a multimeric cytokine receptor may furthercomprise additional amino acid residues (e.g., a polypeptide linker)between the component proteins or polypeptides. A domain linker maycomprise a sequence of amino acids from about 3 to about 20 amino acidslong, from about 5 to 15 about amino acids long, from about 8 to about12 amino acids long, and about 10 amino acids long. One function of alinker is to separate the active protein regions to promote theirindependent bioactivity and permit each region to assume its bioactiveconformation independent of interference from its neighboring structure.

Using the methods discussed herein, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptide fragments or variantsof SEQ ID NO:111, SEQ ID NO:109, SEQ ID NO:5, SEQ ID NO:117 and SEQ IDNO:119 that retain the signal transduction or ligand binding activity.For example, one can make a zcytor17 “soluble receptor” by preparing avariety of polypeptides that are substantially homologous to thecytokine-binding domain (residues 20 (Ala) to 227 (Pro) of SEQ ID NO:111and SEQ ID NO:109; residues 33 (Ala) to 240 (Pro) of SEQ ID NO:5), theextracellular domain (residues 20 (Ala) to 519 (Glu) of SEQ ID NO:111and SEQ ID NO:109; residues 33 (Ala) to 532 (Glu) of SEQ ID NO:5), orallelic variants or species orthologs thereof (e.g., see SEQ ID NO:117and SEQ ID NO:119 and functional fragments thereof as described herein))and retain ligand-binding activity of the wild-type zcytor17 protein.Moreover, variant zcytor17 soluble receptors such as those shown in SEQID NO:113 and SEQ ID NO:115 can be isolated. Such polypeptides mayinclude additional amino acids from, for example, part or all of thetransmembrane and intracellular domains. Such polypeptides may alsoinclude additional polypeptide segments as generally disclosed hereinsuch as labels, affinity tags, and the like.

For any zcytor17 polypeptide, including variants, soluble receptors, andfusion polypeptides or proteins, one of ordinary skill in the art canreadily generate a fully degenerate polynucleotide sequence encodingthat variant using the information set forth in Tables 1 and 2 above.

The zcytor17 multimeric cytokine receptors of the present invention,including full-length polypeptides, biologically active fragments, andfusion polypeptides, can be produced in genetically engineered hostcells according to conventional techniques. Suitable host cells arethose cell types that can be transformed or transfected with exogenousDNA and grown in culture, and include bacteria, fungal cells, andcultured higher eukaryotic cells. Eukaryotic cells, particularlycultured cells of multicellular organisms, are preferred. Techniques formanipulating cloned DNA molecules and introducing exogenous DNA into avariety of host cells are disclosed by Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., eds., CurrentProtocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

The present invention also provides an expression vector comprising anisolated and purified DNA molecule including the following operablylinked elements: a first transcription promoter, a first DNA segmentencoding a polypeptide having at least 90 percent sequence identity withSEQ ID NO:111, and a first transcription terminator; and a secondtranscription promoter, a second DNA segment encoding at least a portionof a class I cytokine receptor, and a second transcription terminator;wherein the polypeptide and the class I cytokine receptor form amultimeric cytokine receptor; and wherein the multimeric cytokinereceptor binds to at least a portion of SEQ ID NO:2. The DNA moleculemay further comprise a secretory signal sequence operably linked to thefirst and second DNA segments. The multimeric cytokine receptor may besoluble and/or may further comprise an affinity tag as described herein.In addition, the multimeric cytokine receptor may antagonize an activityof SEQ ID NO:2 as described herein. The at least at portion of a class Icytokine receptor may comprise portions of SEQ ID NO:7 and/or SEQ IDNO:9, such as, for instance, amino acid residue 28 to amino acid residue429 of SEQ ID NO:7, amino acid residue 35 to amino acid residue 137 ofSEQ ID NO:7, amino acid residue 240 to amino acid residue 342 of SEQ IDNO:7, amino acid residue 348 to amino acid residue 429 of SEQ ID NO:7,amino acid residue 28 to amino acid residue 739 of SEQ ID NO:7, aminoacid residue 28 to amino acid residue 761 of SEQ ID NO:7, amino acidresidue 762 to amino acid residue 979 of SEQ ID NO:7, or combinationsthereof. The present invention also provides a cultured cell containingthe above-described expression vector.

In general, a DNA sequence, for example, encoding a zcytor17 polypeptideis operably linked to other genetic elements required for itsexpression, generally including a transcription promoter and terminator,within an expression vector. The vector will also commonly contain oneor more selectable markers and one or more origins of replication,although those skilled in the art will recognize that within certainsystems selectable markers may be provided on separate vectors, andreplication of the exogenous DNA may be provided by integration into thehost cell genome. Selection of promoters, terminators, selectablemarkers, vectors and other elements is a matter of routine design withinthe level of ordinary skill in the art. Many such elements are describedin the literature and are available through commercial suppliers.

To direct, for example, a zcytor17 polypeptide into the secretorypathway of a host cell, a secretory signal sequence (also known as aleader sequence, prepro sequence or pre sequence) is provided in theexpression vector. The secretory signal sequence may be that ofzcytor17, or may be derived from another secreted protein (e.g., t-PA)or synthesized de novo. The secretory signal sequence is operably linkedto the zcytor17 DNA sequence, i.e., the two sequences are joined in thecorrect reading frame and positioned to direct the newly synthesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe polypeptide of interest, although certain secretory signal sequencesmay be positioned elsewhere in the DNA sequence of interest (see, e.g.,Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.5,143,830).

Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid 1 (Met) toamino acid 19 (Ala) of SEQ ID NO:111 and SEQ ID NO:109, or wherein asecretory signal sequence derived from amino acid 1 (Met) to amino acid32 (Ala) of SEQ ID NO:5, or amino acid 1 (Met) to amino acid 45 (Ala) ofSEQ ID NO:117 or SEQ ID NO:119), or amino acid 28 (Met) to residue 45(Ala) of SEQ ID NO:117 or SEQ ID NO:119), is operably linked to anotherpolypeptide using methods known in the art and disclosed herein. Thesecretory signal sequence contained in the fusion polypeptides of thepresent invention is preferably fused amino-terminally to an additionalpeptide to direct the additional peptide into the secretory pathway.Such constructs have numerous applications known in the art. Forexample, these novel secretory signal sequence fusion constructs candirect the secretion of an active component of a normally non-secretedprotein. Such fusions may be used in vivo or in vitro to direct peptidesthrough the secretory pathway.

The present invention also provides a cultured cell comprising a firstexpression vector comprising a DNA molecule containing the followingoperably linked elements: a transcription promoter, a DNA segmentencoding a polypeptide having at least 90 percent sequence identity withSEQ ID NO:111, and a transcription terminator; and a second expressionvector comprising a transcription promoter, a DNA segment encoding atleast a portion of a class I cytokine receptor, and a transcriptionterminator; wherein the polypeptide and the class I cytokine receptorform a multimeric cytokine receptor. The first and second expressionvectors may further comprise a secretory signal sequence operably linkedto the first and second DNA segments. The multimeric cytokine receptormay be soluble, may be a heterodimer, and/or may further comprise anaffinity tag as described herein. In addition, the multimeric cytokinereceptor may antagonize an activity of SEQ ID NO:2 as described herein.The at least at portion of a class I cytokine receptor may compriseportions of SEQ ID NO:7 and/or SEQ ID NO:9, such as, for instance, aminoacid residue 28 to amino acid residue 429 of SEQ ID NO:7, amino acidresidue 35 to amino acid residue 137 of SEQ ID NO:7, amino acid residue240 to amino acid residue 342 of SEQ ID NO:7, amino acid residue 348 toamino acid residue 429 of SEQ ID NO:7, amino acid residue 28 to aminoacid residue 739 of SEQ ID NO:7, amino acid residue 28 to amino acidresidue 761 of SEQ ID NO:7, amino acid residue 762 to amino acid residue979 of SEQ ID NO:7, or combinations thereof.

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-716, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al, U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Rockville, Md. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus ExpressionSystem: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. etal., Baculovirus Expression Vectors: A Laboratory Manual, New York,Oxford University Press., 1994; and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, Totowa, N.J., HumanaPress, 1995. A second method of making recombinant zcytor17 baculovirusutilizes a transposon-based system described by Luckow (Luckow, V. A, etal., J Virol 67:4566-79, 1993). This system, which utilizes transfervectors, is sold in the Bac-to-Bac™ kit (Life Technologies, Rockville,Md.). This system utilizes a transfer vector, pFastBac1™ (LifeTechnologies) containing a Tn7 transposon to move the DNA encoding thezcytor17 polypeptide into a baculovirus genome maintained in E. coli asa large plasmid called a “bacmid.” See, Hill-Perkins, M. S. and Possee,R. D., J Gen Virol 71:971-6, 1990; Bonning, B. C. et al., J Gen Virol75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, B., J Biol Chem270:1543-9, 1995. In addition, transfer vectors can include an in-framefusion with DNA encoding an epitope tag at the C— or N-terminus of theexpressed zcytor17 polypeptide, for example, a Glu-Glu epitope tag(Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Usinga technique known in the art, a transfer vector containing zcytor17 istransformed into E. coli, and screened for bacmids which contain aninterrupted lacZ gene indicative of recombinant baculovirus. The bacmidDNA containing the recombinant baculovirus genome is isolated, usingcommon techniques, and used to transfect Spodoptera frugiperda cells,e.g., Sf9 cells. Recombinant virus that expresses zcytor17 issubsequently produced. Recombinant viral stocks are made by methodscommonly used in the art.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells.Procedures used are generally described in available laboratory manuals(King, L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et al., ibid.;Richardson, C. D., ibid.). Subsequent purification of the zcytor17polypeptide from the supernatant can be achieved using methods describedherein.

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al, U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanotica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol 132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinantproteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), whichallows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. It is preferred to transform P.methanolica cells by electroporation using an exponentially decaying,pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a zcytor17polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanoticacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanotica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

Within one aspect of the present invention, a zcytor17 multimericcytokine receptor (including transmembrane and intracellular domains) isproduced by a cultured cell, and the cell is used to screen for ligandsfor the receptor, including the natural ligand (SEQ ID NO:2), as well asagonists and antagonists of the natural ligand. To summarize thisapproach, a cDNA or gene encoding the receptor is combined with othergenetic elements required for its expression (e.g., a transcriptionpromoter), and the resulting expression vector is inserted into a hostcell. Cells that express the DNA and produce functional receptor areselected and used within a variety of screening systems.

Mammalian cells suitable for use in expressing the novel receptors ofthe present invention and transducing a receptor-mediated signal includecells that express a β-subunit, such as gp130, and cells that co-expressgp130 and LIF receptor (Gearing et al., EMBO J. 10:2839-2848, 1991;Gearing et al., U.S. Pat. No. 5,284,755). In this regard it is generallypreferred to employ a cell that is responsive to other cytokines thatbind to receptors in the same subfamily, such as IL-6 or LIF, becausesuch cells will contain the requisite signal transduction pathway(s).Preferred cells of this type include BaF3 cells (Palacios and Steinmetz,Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6:4133-4135, 1986), the human TF-1 cell line (ATCC number CRL-2003) andthe DA-1 cell line (Branch et al., Blood 69:1782, 1987; Broudy et al.,Blood 75:1622-1626, 1990). In the alternative, suitable host cells canbe engineered to produce a β-subunit or other cellular component neededfor the desired cellular response. For example, the murine cell lineBaF3 (Palacios and Steinmetz, Cell 41:727-734, 1985; Mathey-Prevot etal., Mol. Cell. Biol. 6: 4133-4135, 1986), a baby hamster kidney (BHK)cell line, or the CTLL-2 cell line (ATCC TIB-214) can be transfected toexpress the mouse gp130 subunit, or mouse gp130 and LIF receptor, inaddition to zcytor17. It is generally preferred to use a host cell andreceptor(s) from the same species, however this approach allows celllines to be engineered to express multiple receptor subunits from anyspecies, thereby overcoming potential limitations arising from speciesspecificity. In the alternative, species homologs of the human receptorcDNA can be cloned and used within cell lines from the same species,such as a mouse cDNA in the BaF3 cell line. Cell lines that aredependent upon one hematopoietic growth factor, such as IL-3, can thusbe engineered to become dependent upon a zcytor17 ligand oranti-zcytor17 antibody.

Cells expressing functional zcytor17 are used within screening assays. Avariety of suitable assays are known in the art. These assays are basedon the detection of a biological response in the target cell. One suchassay is a cell proliferation assay. Cells are cultured in the presenceor absence of a test compound, and cell proliferation is detected by,for example, measuring incorporation of tritiated thymidine or bycolorimetric assay based on the reduction or metabolic breakdown ofAlymar Blue™ (AccuMed, Chicago, Ill.) or3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, J. Immunol. Meth. 65:55-63, 1983). An alternative assay formatuses cells that are further engineered to express a reporter gene. Thereporter gene is linked to a promoter element that is responsive to thereceptor-linked pathway, and the assay detects activation oftranscription of the reporter gene. A preferred promoter element in thisregard is a serum response element, STAT or SRE (see, for example, Shawet al., Cell 56:563-572, 1989). A preferred such reporter gene is aluciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987).Expression of the luciferase gene is detected by luminescence usingmethods known in the art (e.g., Baumgartner et al., J. Biol. Chem.269:19094-29101, 1994; Schenborn and Goiffin, Promega Notes 41:11,1993). Luciferase assay kits are commercially available from, forexample, Promega Corp., Madison, Wis. Target cell lines of this type canbe used to screen libraries of chemicals, cell-conditioned culturemedia, fungal broths, soil samples, water samples, and the like. Forexample, a bank of cell- or tissue-conditioned media samples can beassayed on a target cell to identify cells that produce ligand. Positivecells are then used to produce a cDNA library in a mammalian cellexpression vector, which is divided into pools, transfected into hostcells, and expressed. Media samples from the transfected cells are thenassayed, with subsequent division of pools, retransfection,subculturing, and re-assay of positive cells to isolate a clonal cellline expressing the ligand. Media samples conditioned by kidney, liver,spleen, thymus, other lymphoid tissues, or T-cells are preferred sourcesof ligand for use in screening procedures.

The present invention also provides a method of producing a multimericcytokine receptor. The method includes culturing a cell as describedherein, and isolating the multimeric cytokine receptor produced by thecell.

The present invention also provides a method for detecting a multiplecytokine receptor ligand in a test sample. The method includescontacting the test sample with a multimeric cytokine receptorcomprising a polypeptide comprising amino acid residue 20 to amino acidresidue 227 of SEQ ID NO:111 or SEQ ID NO:109 (amino acid residue 33 toamino acid residue 240 of SEQ ID NO:5), and at least a portion of atleast one class I cytokine receptor; and detecting the binding of themultimer cytokine receptor to the ligand in the test sample. The atleast a portion of at least one class I cytokine receptor can include,for example, a portion of SEQ ID NO:9 and/or a portion of SEQ ID NO:7,such as, for instance, amino acid residue 28 to amino acid residue 429of SEQ ID NO:7, amino acid residue 35 to amino acid residue 137 of SEQID NO:7, amino acid residue 240 to amino acid residue 342 of SEQ IDNO:7, amino acid residue 348 to amino acid residue 429 of SEQ ID NO:7,amino acid residue 28 to amino acid residue 739 of SEQ ID NO:7, and/orcombinations thereof.

A natural ligand for a zcytor17 multimeric cytokine receptor of thepresent invention can also be identified by mutagenizing acytokine-dependent cell line expressing zcytor17 and culturing it underconditions that select for autocrine growth. See WIPO publication WO95/21930. Within a typical procedure, cells expressing zcytor17 aremutagenized, such as with EMS. The cells are then allowed to recover inthe presence of the required cytokine, then transferred to a culturemedium lacking the cytokine. Surviving cells are screened for theproduction of a ligand for zcytor17 multimeric cytokine receptor, suchas by adding soluble receptor polypeptide comprising the zcytor17cytokine-binding domain and at least a portion of a class I cytokinereceptor, such as the cytokine-binding domain of OSMRbeta (SEQ ID NO:7)and/or WSX-1 (SEQ ID NO:9), as described herein to the culture medium tocompete against the ligand or by assaying conditioned media on wild-typecells compared to transfected cells expressing the zcytor17 multimericcytokine receptor. Preferred cell lines for use within this methodinclude cells that are transfected to express gp130 or gp130 incombination with LIF receptor. Preferred such host cell lines includetransfected CTLL-2 cells (Gillis and Smith, Nature 268:154-156, 1977)and transfected BaF3 cells.

Moreover, a secretion trap method employing zcytor17 soluble multimericcytokine receptor can be used to isolate a zcytor17 ligand, such as SEQID NO:2 (Aldrich, et al, Cell 87: 1161-1169, 1996). A cDNA expressionlibrary prepared from a known or suspected ligand source is transfectedinto COS-7 cells. The cDNA library vector generally has an SV40 originfor amplification in COS-7 cells, and a CMV promoter for highexpression. The transfected COS-7 cells are grown in a monolayer andthen fixed and permeabilized. Tagged or biotin-labeled zcytor17 solublemultimeric cytokine receptor, described herein, is then placed incontact with the cell layer and allowed to bind cells in the monolayerthat express an anti-complementary molecule, i.e., a zcytor17 ligand. Acell expressing a ligand will thus be bound with receptor molecules. Ananti-tag antibody (anti-Ig for Ig fusions, M2 or anti-FLAG forFLAG-tagged fusions, streptavidin, anti-Glu-Glu tag, and the like) whichis conjugated with horseradish peroxidase (HRP) is used to visualizethese cells to which the tagged or biotin-labeled zcytor17 solublemultimeric cytokine receptor has bound. The HRP catalyzes deposition ofa tyramide reagent, for example, tyramide-FITC. A commercially-availablekit can be used for this detection (for example, Renaissance TSA-Direct™Kit; NEN Life Science Products, Boston, Mass.). Cells which expresszcytor17 multimeric cytokine receptor ligand will be identified underfluorescence microscopy as green cells and picked for subsequent cloningof the ligand using procedures for plasmid rescue as outlined inAldrich, et al, supra., followed by subsequent rounds of secretion trapassay, or conventional screening of cDNA library pools, until singleclones are identified.

As a multimeric receptor complex, the activity of zcytor17 polypeptidecan be measured by a silicon-based biosensor microphysiometer whichmeasures the extracellular acidification rate or proton excretionassociated with receptor binding and subsequent physiologic cellularresponses. An exemplary device is the Cytosensor™ Microphysiometermanufactured by Molecular Devices, Sunnyvale, Calif. A variety ofcellular responses, such as cell proliferation, ion transport, energyproduction, inflammatory response, regulatory and receptor activation,and the like, can be measured by this method. See, for example,McConnell, H. M. et al., Science 257:1906-1912, 1992; Pitchford, S. etal., Meth. Enzymol. 228:84-108, 1997; Arimilli, S. et al., J. Immunol.Meth. 212:49-59, 1998; Van Liefde, I. Et al., Eur. J. Pharmacol.346:87-95, 1998. The microphysiometer can be used for assayingeukaryotic, prokaryotic, adherent or non-adherent cells. By measuringextracellular acidification changes in cell media over time, themicrophysiometer directly measures cellular responses to variousstimuli, including agonists, ligands, or antagonists of the zcytor17polypeptide. Preferably, the microphysiometer is used to measureresponses of a zcytor17-expressing eukaryotic cell, compared to acontrol eukaryotic cell that does not express zcytor17 polypeptide.Zcytor17-expressing eukaryotic cells comprise cells into which zcytor17has been transfected or infected via adenovirus vector, and the like, asdescribed herein, creating a cell that is responsive tozcytor17-modulating stimuli, or are cells naturally expressing zcytor17,such as zcytor17-expressing cells derived from lymphoid, spleen, thymustissue or PBLs. Differences, measured by an increase or decrease inextracellular acidification, in the response of cells expressingzcytor17, relative to a control, are a direct measurement ofzcytor17-modulated cellular responses. Moreover, such zcytor17-modulatedresponses can be assayed under a variety of stimuli. Also, using themicrophysiometer, there is provided a method of identifying agonists andantagonists of zcytor17 multimeric cytokine receptor, comprisingproviding cells expressing a zcytor17 multimeric cytokine receptor,culturing a first portion of the cells in the absence of a testcompound, culturing a second portion of the cells in the presence of atest compound, and detecting an increase or a decrease in a cellularresponse of the second portion of the cells as compared to the firstportion of the cells. Antagonists and agonists, including the naturalligand for zcytor17 multimeric cytokine receptor, can be rapidlyidentified using this method.

A zcytor17 multimeric cytokine receptor can be expressed as a fusionwith an immunoglobulin heavy chain constant region, typically an F_(c)fragment, which contains two constant region domains and lacks thevariable region. Methods for preparing such fusions are disclosed inU.S. Pat. Nos. 5,155,027 and 5,567,584. Such fusions are typicallysecreted as multimeric molecules wherein the F_(c) portions aredisulfide bonded to each other and two non-Ig polypeptides are arrayedin closed proximity to each other. Fusions of this type can be used forexample, for dimerization, increasing stability and in vivo half-life,to affinity purify ligand, as in vitro assay tool or antagonist. For usein assays, the chimeras are bound to a support via the F_(c) region andused in an ELISA format.

Additional assays provided by the present invention include the use ofhybrid receptor polypeptides. These hybrid polypeptides fall into twogeneral classes. Within the first class, the intracellular domain ofzcytor17, comprising approximately residues 544 (Lys) to 732 (Val) ofSEQ ID NO:111, residues 544 (Lys) to 649 (Ile) of SEQ ID NO:109, orresidues 557 (Lys) to 662 (Ile) of SEQ ID NO:5, or residues 551 (Lys) to662 (Cys) of SEQ ID NO:117 is joined to the ligand-binding domain of asecond receptor. It is preferred that the second receptor be ahematopoietic cytokine receptor, such as, for instance, mp1 receptor(Souyri et al., Cell 63:1137-1147, 1990). The hybrid receptor willfurther comprise a transmembrane domain, which may be derived fromeither receptor. A DNA construct encoding the hybrid receptor is theninserted into a host cell. Cells expressing the hybrid receptor arecultured in the presence of a ligand for the binding domain and assayedfor a response. This system provides a means for analyzing signaltransduction mediated by zcytor17 while using readily available ligands.This system can also be used to determine if particular cell lines arecapable of responding to signals transduced by zcytor17. A second classof hybrid receptor polypeptides comprise the extracellular(ligand-binding) domain (approximately residues 20 (Ala) to 519 (Glu) ofSEQ ID NO:111 and SEQ ID NO:109; approximately residues 33 (Ala) to 532(Glu) of SEQ ID NO:5) or cytokine-binding domain of zcytor17(approximately residues 20 (Ala) to 227 (Pro) of SEQ ID NO:111 and SEQID NO:109; or approximately residues 33 (Ala) to 240 (Pro) of SEQ IDNO:5; approximately residues 46 (Val) to 533 (Glu) of SEQ ID NO:117; orapproximately residues 46 (Val) to 533 (Trp) of SEQ ID NO:119) with acytoplasmic domain of a second receptor, preferably a cytokine receptor,and a transmembrane domain. The transmembrane domain may be derived fromeither receptor. Hybrid receptors of this second class are expressed incells known to be capable of responding to signals transduced by thesecond receptor. Together, these two classes of hybrid receptors enablethe use of a broad spectrum of cell types within receptor-based assaysystems.

The expression of WSX-1 is strongest in thymus, spleen, PBL, and lymphnode, as well as increased expression observed for activated T-cells.The tissue distribution for OSMRbeta is described as very broad. Thetissue distribution of these three receptors suggests that a target forzcytor17lig is hematopoietic lineage cells, in particular T-cells,monocytes/macrophages and lymphoid progenitor cells and lymphoid cells.Other known four-helical-bundle cytokines that act on lymphoid cellsinclude IL-2, IL-4, IL-7, and IL-15. For a review of four-helical-bundlecytokines, see, Nicola et al., Advances in Protein Chemistry 52:1-65,1999 and Kelso, A., Immunol. Cell. Biol. 76:300-317, 1998.

Conditioned media (CM) from CD3+ selected, PMA/Ionomycin-stimulatedhuman peripheral blood cells supported the growth of BaF3 cells thatexpressed the zcytor17 receptor, OSMRbeta and WSX-1 receptor and wereotherwise dependent on IL-3. Conditioned medias from cells that werenot: 1) PMA/Ionomycin-stimulated; or were not: 2) CD3 selected (with orwithout PMA/Ionomycin stimulation) did not support the growth of Baf3cells expressing zcytor17, OSMRbeta and WSX-1(BaF3/zcytor17/WSX-1/OSMRbeta) receptor-expressing cells. Controlexperiments demonstrated that this proliferative activity was notattributable to other known growth factors, and that the ability of suchconditioned media to stimulate proliferation of zcytor17/WSX-1/OSMRbetareceptor-expressing cells could be neutralized by a soluble form of thezcytor17 receptor.

Conditioned-media from CD3+ selected cells activated with PMA/Ionomycinalso supported growth of BaF3 cells that expressed the zcytor17 receptorand OSMRbeta receptor (zcytor17/OSMRbeta), while BaF3 cells expressingonly zcytor17 receptor and WSX-1 receptor (zcytor17/WSX-1), orcontaining only the OSMRbeta receptor, were not stimulated by thisconditioned-media.

Proliferation of zcytor17/WSX-1/OSMRbeta receptor-expressing BaF3 cellsexposed to CM from CD3+ selected, PMA/Ionomycin-stimulated humanperipheral blood cells were identified by visual inspection of thecultures and/or by proliferation assay. Many suitable proliferationassays are known in the art, and include assays for reduction of a dyesuch as AlamarBlue™ (AccuMed International, Inc. Westlake, Ohio),3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Mosman,J. Immunol. Meth. 65: 55-63, 1983);3,(4,5dimethylthiazol-2yl)-5-3-carboxymethoxyphenyl-2H-tetrazolium;2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide; and cyanoditolyl-tetrazolium chloride (which are commerciallyavailable from Polysciences, Inc., Warrington, Pa.); mitogenesis assays,such as measurement of incorporation of ³H-thymidine; dye exclusionassays using, for example, naphthalene black or trypan blue; dye uptakeusing diacetyl fluorescein; and chromium release. See, in general,Freshney, Culture of Animal Cells: A Manual of Basic Technique, 3rd ed.,Wiley-Liss, 1994, which is incorporated herein by reference.

A cDNA library was prepared from CD3+ selected, PMA- andIonomycin-stimulated primary human peripheral blood cells. The CD3+selected, PMA- and Ionomycin-stimulated human peripheral blood cellscDNA library was divided into pools containing multiple cDNA moleculesand was transfected into a host cell line, for example, BHK 570 cells(ATCC accession no. 10314). The transfected host cells were cultured ina medium that did not contain exogenous growth factors (e.g., 5% FBS)and conditioned medium was collected. The conditioned media were assayedfor the ability to stimulate proliferation of BaF3 cells transfectedwith the zcytor17, WSX-1, and OSMRbeta receptors. cDNA pools producingconditioned medium that stimulated BaF3/zcytor17/WSX-1/OSMRbeta receptorcells were identified. This pooled plasmid cDNA was electroporated intoE. coli. cDNA was isolated from single colonies and transfectedindividually into BHK 570 cells. Positive clones were identified by apositive result in the BaF3/zcytor17/WSX-1/OSMRbeta receptorproliferation assay, and the activity was confirmed by neutralization ofproliferation using the soluble zcytor17 receptor.

In view of the tissue distribution observed for zcytor17 receptoragonists (including the natural zcytor17lig/substrate/cofactor/etc.)and/or antagonists have enormous potential in both in vitro and in vivoapplications. Compounds identified as zcytor17lig agonists are usefulfor expansion, proliferation, activation, differentiation, and/orinduction or inhibition of specialized cell functions of cells involvedin homeostasis of hematopoiesis and immune function. For example,zcytor17lig and agonist compounds are useful as components of definedcell culture media, and may be used alone or in combination with othercytokines and hormones to replace serum that is commonly used in cellculture. Agonists are thus useful in specifically promoting the growthand/or development of T-cells, B-cells, monocytes/macrophages, NK cells,cytotoxic lymphocytes, and other cells of the lymphoid and myeloidlineages in culture.

Antagonists are also useful as research reagents for characterizingsites of ligand-receptor interaction. Antagonists are useful to inhibitexpansion, proliferation, activation, and/or differentiation of cellsinvolved in regulating hematopoiesis. Inhibitors of zcytor17lig activity(zcytor17lig antagonists) include anti-zcytor17lig antibodies andsoluble multimeric cytokine receptors, as well as other peptidic andnon-peptidic agents (including ribozymes).

A zcytor17lig-binding protein, such as a multimeric cytokine receptor ofthe present invention, can also be used for purification of ligand. Themultimeric cytokine receptor is immobilized on a solid support, such asbeads of agarose, cross-linked agarose, glass, cellulosic resins,silica-based resins, polystyrene, cross-linked polyacrylamide, or likematerials that are stable under the conditions of use. Methods forlinking polypeptides to solid supports are known in the art, and includeamine chemistry, cyanogen bromide activation, N-hydroxysuccinimideactivation, epoxide activation, sulfhydryl activation, and hydrazideactivation. The resulting medium will generally be configured in theform of a column, and fluids containing ligand are passed through thecolumn one or more times to allow ligand to bind to the receptorpolypeptide. The ligand is then eluted using changes in saltconcentration, chaotropic agents (guanidine HCR), or pH to disruptligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument (BIAcore,Pharmacia Biosensor, Piscataway, N.J.) may be advantageously employed.Such receptor, antibody, member of a complement/anti-complement pair orfragment is immobilized onto the surface of a receptor chip. Use of thisinstrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40(1991) and Cunningham and Wells, J. Mol. Biol. 234:554-63 (1993). Areceptor, antibody, member or fragment is covalently attached, usingamine or sulfhydryl chemistry, to dextran fibers that are attached togold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.Alternatively, ligand/receptor binding can be analyzed using SELDI(TM)technology (Ciphergen, Inc., Palo Alto, Calif.).

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-72 (1949)) and calorimetric assays (Cunningham et al., Science253:545-48 (1991); and Cunningham et al., Science 245:821-25 (1991)).

The present invention also provides an antibody that specifically bindsto a polypeptide or at least at portion of a multimeric cytokinereceptor as described herein.

Zcytor17 multimeric cytokine receptors can also be used to prepareantibodies that bind to epitopes, peptides or polypeptides thereof. Themultimeric cytokine receptor or a fragment thereof serves as an antigen(immunogen) to inoculate an animal and elicit an immune response. One ofskill in the art would recognize that antigenic, epitope-bearingpolypeptides may contain a sequence of at least 6, preferably at least9, and more preferably at least 15 to about 30 contiguous amino acidresidues of a polypeptide(s) of the multimeric cytokine receptor, suchas zcytor17 (SEQ ID NO:111), OSMRbeta (SEQ ID NO:7), and/or WSX-1 (SEQID NO:9). Polypeptides comprising a larger portion of a multimericcytokine receptor, i.e., from 30 to 100 residues up to the entire lengthof the amino acid sequence are included. Antigens or immunogenicepitopes can also include attached tags, adjuvants, carriers andvehicles, as described herein.

Antibodies from an immune response generated by inoculation of an animalwith these antigens can be isolated and purified as described herein.Methods for preparing and isolating polyclonal and monoclonal antibodiesare well known in the art. See, for example, Current Protocols inImmunology, Cooligan, et al. (eds.), National Institutes of Health, JohnWiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; andHurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, Fla., 1982.

As would be evident to one of ordinary skill in the art, polyclonalantibodies can be generated from inoculating a variety of warm-bloodedanimals such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, and rats with a multimeric cytokine receptor or a fragmentthereof. The immunogenicity of a multimeric cytokine receptor may beincreased through the use of an adjuvant, such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Multimericcytokine receptors useful for immunization also include fusionpolypeptides, such as fusions of zcytor17, OSMRbeta, and/or WSX-1, or aportion thereof with an immunoglobulin polypeptide or with maltosebinding protein. The polypeptide immunogen may be a full-length moleculeor a portion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Moreover, human antibodies can beproduced in transgenic, non-human animals that have been engineered tocontain human immunoglobulin genes as disclosed in WIPO Publication WO98/24893. It is preferred that the endogenous immunoglobulin genes inthese animals be inactivated or eliminated, such as by homologousrecombination.

Antibodies are considered to be specifically binding if: 1) they exhibita threshold level of binding activity, and 2) they do not significantlycross-react with related polypeptide molecules. A threshold level ofbinding is determined if anti-multimeric cytokine receptor antibodiesherein bind to a multimeric cytokine receptor, peptide or epitope withan affinity at least 10-fold greater than the binding affinity tocontrol (non-multimeric cytokine receptor) protein. It is preferred thatthe antibodies exhibit a binding affinity (K_(a)) of 10⁶ M⁻¹ or greater,preferably 10⁷ M⁻¹ or greater, more preferably 10⁸ M⁻¹ or greater, andmost preferably 10⁹ M⁻¹ or greater. The binding affinity of an antibodycan be readily determined by one of ordinary skill in the art, forexample, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51:660-672 (1949)).

Whether anti-multimeric cytokine receptor antibodies do notsignificantly cross-react with related polypeptide molecules is shown,for example, by the antibody detecting zcytor17 multimeric cytokinereceptor but not known related polypeptides using a standard Westernblot analysis (Ausubel et al., ibid.). Examples of known relatedpolypeptides are those disclosed in the prior art, such as knownorthologs, and paralogs, and similar known members of a protein family.Screening can also be done using non-human multimeric cytokine receptor,and multimeric cytokine receptor mutant polypeptides. Moreover,antibodies can be “screened against” known related polypeptides, toisolate a population that specifically binds to the multimeric cytokinereceptor. For example, antibodies raised to multimeric cytokine receptorare adsorbed to related polypeptides adhered to insoluble matrix;antibodies specific to multimeric cytokine receptor will flow throughthe matrix under the proper buffer conditions. Screening allowsisolation of polyclonal and monoclonal antibodies non-crossreactive toknown closely related polypeptides (Antibodies: A Laboratory Manual,Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988;Current Protocols in Immunology, Cooligan, et al. (eds.), NationalInstitutes of Health, John Wiley and Sons, Inc., 1995). Screening andisolation of specific antibodies is well known in the art. See,Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al.,Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principles andPractice, Goding, J. W. (eds.), Academic Press Ltd., 1996; Benjamin etal., Ann. Rev. Immunol. 2: 67-101, 1984. Specifically bindinganti-multimeric cytokine receptor antibodies can be detected by a numberof methods in the art, and disclosed below.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which bind to multimeric cytokine receptor proteins orpolypeptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutantmultimeric cytokine receptor protein or polypeptide.

Within another aspect the present invention provides an antibodyproduced by the method as disclosed above, wherein the antibody binds toat least a portion of a multimer cytokine receptor comprising at least aportion of SEQ ID NO:111, SEQ ID NO:109, or SEQ ID NO:5. In oneembodiment, the antibody disclosed above specifically binds to apolypeptide shown in SEQ ID NO:111, SEQ ID NO:109, or SEQ ID NO:5. Inanother embodiment, the antibody can be a monoclonal antibody or apolyclonal antibody.

Antibodies to multimeric cytokine receptor may be used for tagging cellsthat express multimeric cytokine receptor; for isolating multimericcytokine receptor by affinity purification; for diagnostic assays fordetermining circulating levels of multimeric cytokine receptor; fordetecting or quantitating soluble multimeric cytokine receptor as amarker of underlying pathology or disease; in analytical methodsemploying FACS; for screening expression libraries; for generatinganti-idiotypic antibodies; and as neutralizing antibodies or asantagonists to block multimeric cytokine receptor activity in vitro andin vivo. Suitable direct tags or labels include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like; indirect tags or labels mayfeature use of biotin-avidin or other complement/anti-complement pairsas intermediates. Antibodies herein may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.Moreover, antibodies to multimeric cytokine receptor or fragmentsthereof may be used in vitro to detect denatured multimeric cytokinereceptor or fragments thereof in assays, for example, Western Blots orother assays known in the art.

Suitable detectable molecules may be directly or indirectly attached tothe multimeric cytokine receptor or antibody, and include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like. Suitablecytotoxic molecules may be directly or indirectly attached to thepolypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria, toxin, saporin, Pseudomonas exotoxin, ricin, abrinand the like), as well as therapeutic radionuclides, such as iodine-131,rhenium-188 or yttrium-90 (either directly attached to the polypeptideor antibody, or indirectly attached through means of a chelating moiety,for instance). Multimeric cytokine receptors or antibodies may also beconjugated to cytotoxic drugs, such as adriamycin. For indirectattachment of a detectable or cytotoxic molecule, the detectable orcytotoxic molecule can be conjugated with a member of acomplementary/anticomplementary pair, where the other member is bound tothe polypeptide or antibody portion. For these purposes,biotin/streptavidin is an exemplary complementary/anticomplementarypair.

A soluble multimeric cytokine receptor can also act as zcytor17lig“antagonists” to block zcytor17lig binding and signal transduction invitro and in vivo. These anti-zcytor17lig binding proteins would beuseful for inhibiting zcytor17lig activity or protein-binding.

Polypeptide-toxin fusion proteins or antibody-toxin fusion proteins canbe used for targeted cell or tissue inhibition or ablation (forinstance, to treat cancer cells or tissues). Alternatively, if thepolypeptide has multiple functional domains (i.e., an activation domainor a receptor binding domain, plus a targeting domain), a fusion proteinincluding only the targeting domain may be suitable for directing adetectable molecule, a cytotoxic molecule or a complementary molecule toa cell or tissue type of interest. In instances where the domain onlyfusion protein includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

Moreover, inflammation is a protective response by an organism to fendoff an invading agent. Inflammation is a cascading event that involvesmany cellular and humoral mediators. On one hand, suppression ofinflammatory responses can leave a host immunocompromised; however, ifleft unchecked, inflammation can lead to serious complications includingchronic inflammatory diseases (e.g., rheumatoid arthritis, multiplesclerosis, inflammatory bowel disease and the like), septic shock andmultiple organ failure. Importantly, these diverse disease states sharecommon inflammatory mediators. The collective diseases that arecharacterized by inflammation have a large impact on human morbidity andmortality. Therefore it is clear that anti-inflammatory antibodies andbinding polypeptides, such as anti-zcytor17lig antibodies and bindingpolypeptides described herein, could have crucial therapeutic potentialfor a vast number of human and animal diseases, from asthma and allergyto autoimmunity and septic shock. As such, use of anti-inflammatory antizcytor17lig antibodies and binding polypeptides described herein can beused therapeutically as zcytor17lig antagonists described herein,particularly in diseases such as arthritis, endotoxemia, inflammatorybowel disease, psoriasis, related disease and the like.

Arthritis

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions which would benefit from the therapeutic use ofanti-inflammatory antibodies and binding polypeptides, such asanti-zcytor17lig antibodies and binding polypeptides of the presentinvention. For example, rheumatoid arthritis (RA) is a systemic diseasethat affects the entire body and is one of the most common forms ofarthritis. It is characterized by the inflammation of the membranelining the joint, which causes pain, stiffness, warmth, redness andswelling. Inflammatory cells release enzymes that may digest bone andcartilage. As a result of rheumatoid arthritis, the inflamed jointlining, the synovium, can invade and damage bone and cartilage leadingto joint deterioration and severe pain amongst other physiologiceffects. The involved joint can lose its shape and alignment, resultingin pain and loss of movement.

Rheumatoid arthritis (RA) is an immune-mediated disease particularlycharacterized by inflammation and subsequent tissue damage leading tosevere disability and increased mortality. A variety of cytokines areproduced locally in the rheumatoid joints. Numerous studies havedemonstrated that IL-1 and TNF-alpha, two prototypic pro-inflammatorycytokines, play an important role in the mechanisms involved in synovialinflammation and in progressive joint destruction. Indeed, theadministration of TNF-alpha and IL-1 inhibitors in patients with RA hasled to a dramatic improvement of clinical and biological signs ofinflammation and a reduction of radiological signs of bone erosion andcartilage destruction. However, despite these encouraging results, asignificant percentage of patients do not respond to these agents,suggesting that other mediators are also involved in the pathophysiologyof arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149, 2002). Oneof those mediators could be zcytor17lig, and as such a molecule thatbinds or inhibits zcytor17lig, such as anti zcytor17lig antibodies orbinding partners, could serve as a valuable therapeutic to reduceinflammation in rheumatoid arthritis, and other arthritic diseases.

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop chronic inflammatory arthritis that closely resembles humanrheumatoid arthritis. Since CIA shares similar immunological andpathological features with RA, this makes it an ideal model forscreening potential human anti-inflammatory compounds. The CIA model isa well-known model in mice that depends on both an immune response, andan inflammatory response, in order to occur. The immune responsecomprises the interaction of B-cells and CD4+ T-cells in response tocollagen, which is given as antigen, and leads to the production ofanti-collagen antibodies. The inflammatory phase is the result of tissueresponses from mediators of inflammation, as a consequence of some ofthese antibodies cross-reacting to the mouse's native collagen andactivating the complement cascade. An advantage in using the CIA modelis that the basic mechanisms of pathogenesis are known. The relevantT-cell and B-cell epitopes on type II collagen have been identified, andvarious immunological (e.g., delayed-type hypersensitivity andanti-collagen antibody) and inflammatory (e.g., cytokines, chemokines,and matrix-degrading enzymes) parameters relating to immune-mediatedarthritis have been determined, and can thus be used to assess testcompound efficacy in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20,1999; Williams et al., Immunol. 89:9784-788, 1992; Myers et al., LifeSci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959, 1995).

The administration of soluble zcytor17 comprising polypeptides(including heterodimeric and multimeric receptors described herein),such as zcytor17-Fc4 or other zcytor17 soluble and fusion proteins tothese CIA model mice was used to evaluate the use of zcytor17 toameliorate symptoms and alter the course of disease. As a molecule thatmodulates immune and inflammatory response, zcytor17lig, may induceproduction of SAA, which is implicated in the pathogenesis of rheumatoidarthritis, zcytor17lig antagonists may reduce SAA activity in vitro andin vivo, the systemic or local administration of zcytor17lig antagonistssuch as anti-zcytor17lig antibodies or binding partners, zcytor17comprising polypeptides (including heterodimeric and multimericreceptors described herein), such as zcytor17-Fc4 or other zcytor17soluble and fusion proteins can potentially suppress the inflammatoryresponse in RA. Other potential therapeutics include zcytor17polypeptides, soluble heterodimeric and multimeric receptorpolypeptides, or anti zcytor17lig antibodies or binding partners of thepresent invention, and the like.

Endotoxemia

Endotoxemia is a severe condition commonly resulting from infectiousagents such as bacteria and other infectious disease agents, sepsis,toxic shock syndrome, or in immunocompromised patients subjected toopportunistic infections, and the like. Therapeutically useful ofanti-inflammatory antibodies and binding polypeptides, such asanti-zcytor17lig antibodies and binding polypeptides of the presentinvention, could aid in preventing and treating endotoxemia in humansand animals. Other potential therapeutics include zcytor17 polypeptides,soluble heterodimeric and multimeric receptor polypeptides, or antizcytor17lig antibodies or binding partners of the present invention, andthe like, could serve as a valuable therapeutic to reduce inflammationand pathological effects in endotoxemia.

Lipopolysaccharide (LPS) induced endotoxemia engages many of theproinflammatory mediators that produce pathological effects in theinfectious diseases and LPS induced endotoxemia in rodents is a widelyused and acceptable model for studying the pharmacological effects ofpotential pro-inflammatory or immunomodulating agents. LPS, produced ingram-negative bacteria, is a major causative agent in the pathogenesisof septic shock (Glausner et al., Lancet 338:732, 1991). A shock-likestate can indeed be induced experimentally by a single injection of LPSinto animals. Molecules produced by cells responding to LPS can targetpathogens directly or indirectly. Although these biological responsesprotect the host against invading pathogens, they may also cause harm.Thus, massive stimulation of innate immunity, occurring as a result ofsevere Gram-negative bacterial infection, leads to excess production ofcytokines and other molecules, and the development of a fatal syndrome,septic shock syndrome, which is characterized by fever, hypotension,disseminated intravascular coagulation, and multiple organ failure(Dumitru et al. Cell 103:1071-1083, 2000).

These toxic effects of LPS are mostly related to macrophage activationleading to the release of multiple inflammatory mediators. Among thesemediators, TNF appears to play a crucial role, as indicated by theprevention of LPS toxicity by the administration of neutralizinganti-TNF antibodies (Beutler et al., Science 229:869, 1985). It is wellestablished that lug injection of E. coli LPS into a C57B1/6 mouse willresult in significant increases in circulating IL-6, TNF-alpha, IL-1,and acute phase proteins (for example, SAA) approximately 2 hours postinjection. The toxicity of LPS appears to be mediated by these cytokinesas passive immunization against these mediators can result in decreasedmortality (Beutler et al., Science 229:869, 1985). The potentialimmunointervention strategies for the prevention and/or treatment ofseptic shock include anti-TNF mAb, IL-1 receptor antagonist, LIF, IL-10,and G-CSF. Since LPS induces the production of pro-inflammatory factorspossibly contributing to the pathology of endotoxemia, theneutralization of zcytor17lig activity, SAA or other pro-inflammatoryfactors by antagonizing zcytor17lig polypeptide can be used to reducethe symptoms of endotoxemia, such as seen in endotoxic shock. Otherpotential therapeutics include zcytor17 polypeptides, solubleheterodimeric and multimeric receptor polypeptides, or anti-zcytor17ligantibodies or binding partners of the present invention, and the like.

Inflammatory Bowel Disease. IBD

In the United States approximately 500,000 people suffer fromInflammatory Bowel Disease (IBD) which can affect either colon andrectum (Ulcerative colitis) or both, small and large intestine (Crohn'sDisease). The pathogenesis of these diseases is unclear, but theyinvolve chronic inflammation of the affected tissues. Potentialtherapeutics include zcytor17 polypeptides, soluble heterodimeric andmultimeric receptor polypeptides, or anti-zcytor17lig antibodies orbinding partners of the present invention, and the like, could serve asa valuable therapeutic to reduce inflammation and pathological effectsin IBD and related diseases.

Ulcerative colitis (UC) is an inflammatory disease of the largeintestine, commonly called the colon, characterized by inflammation andulceration of the mucosa or innermost lining of the colon. Thisinflammation causes the colon to empty frequently, resulting indiarrhea. Symptoms include loosening of the stool and associatedabdominal cramping, fever and weight loss. Although the exact cause ofUC is unknown, recent research suggests that the body's natural defensesare operating against proteins in the body which the body thinks areforeign (an “autoimmune reaction”). Perhaps because they resemblebacterial proteins in the gut, these proteins may either instigate orstimulate the inflammatory process that begins to destroy the lining ofthe colon. As the lining of the colon is destroyed, ulcers formreleasing mucus, pus and blood. The disease usually begins in the rectalarea and may eventually extend through the entire large bowel. Repeatedepisodes of inflammation lead to thickening of the wall of the intestineand rectum with scar tissue. Death of colon tissue or sepsis may occurwith severe disease. The symptoms of ulcerative colitis vary in severityand their onset may be gradual or sudden. Attacks may be provoked bymany factors, including respiratory infections or stress.

Although there is currently no cure for UC available, treatments arefocused on suppressing the abnormal inflammatory process in the colonlining. Treatments including corticosteroids immunosuppressives (eg.azathioprine, mercaptopurine, and methotrexate) and aminosalicytates areavailable to treat the disease. However, the long-term use ofimmunosuppressives such as corticosteroids and azathioprine can resultin serious side effects including thinning of bones, cataracts,infection, and liver and bone marrow effects. In the patients in whomcurrent therapies are not successful, surgery is an option. The surgeryinvolves the removal of the entire colon and the rectum.

There are several animal models that can partially mimic chroniculcerative colitis. The most widely used model is the2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis model,which induces chronic inflammation and ulceration in the colon. WhenTNBS is introduced into the colon of susceptible mice via intra-rectalinstillation, it induces T-cell mediated immune response in the colonicmucosa, in this case leading to a massive mucosal inflammationcharacterized by the dense infiltration of T-cells and macrophagesthroughout the entire wall of the large bowel. Moreover, thishistopathologic picture is accompanies by the clinical picture ofprogressive weight loss (wasting), bloody diarrhea, rectal prolapse, andlarge bowel wall thickening (Neurath et al. Intern. Rev. Immunol.19:51-62, 2000).

Another colitis model uses dextran sulfate sodium (DSS), which inducesan acute colitis manifested by bloody diarrhea, weight loss, shorteningof the colon and mucosal ulceration with neutrophil infiltration.DSS-induced colitis is characterized histologically by infiltration ofinflammatory cells into the lamina propria, with lymphoid hyperplasia,focal crypt damage, and epithelial ulceration. These changes are thoughtto develop due to a toxic effect of DSS on the epithelium and byphagocytosis of lamina propria cells and production of TNF-alpha andIFN-gamma. Despite its common use, several issues regarding themechanisms of DSS about the relevance to the human disease remainunresolved. DSS is regarded as a T cell-independent model because it isobserved in T cell-deficient animals such as SCID mice.

The administration of anti-zcytor17lig antibodies or binding partners,soluble zcytor17 comprising polypeptides (including heterodimeric andmultimeric receptors), such as zcytor17-Fc4 or other zcytor17 solubleand fusion proteins to these TNBS or DSS models can be used to evaluatethe use of zcytor17lig antagonists to ameliorate symptoms and alter thecourse of gastrointestinal disease. Zcytor17lig may play a role in theinflammatory response in colitis, and the neutralization of zcytor17ligactivity by administrating zcytor17lig antagonists is a potentialtherapeutic approach for IBD. Other potential therapeutics includezcytor17 polypeptides, soluble heterodimeric and multimeric receptorpolypeptides, or anti-zcytor17lig antibodies or binding partners of thepresent invention, and the like.

Psoriasis

Psoriasis is a chronic skin condition that affects more than sevenmillion Americans. Psoriasis occurs when new skin cells grow abnormally,resulting in inflamed, swollen, and scaly patches of skin where the oldskin has not shed quickly enough. Plaque psoriasis, the most commonform, is characterized by inflamed patches of skin (“lesions”) toppedwith silvery white scales. Psoriasis may be limited to a few plaques orinvolve moderate to extensive areas of skin, appearing most commonly onthe scalp, knees, elbows and trunk. Although it is highly visible,psoriasis is not a contagious disease. The pathogenesis of the diseasesinvolves chronic inflammation of the affected tissues. Zcytor17polypeptides, soluble heterodimeric and multimeric receptorpolypeptides, or anti-zcytor17lig antibodies or binding partners of thepresent invention, and the like, could serve as a valuable therapeuticto reduce inflammation and pathological effects in psoriasis, otherinflammatory skin diseases, skin and mucosal allergies, and relateddiseases.

Psoriasis is a T-cell mediated inflammatory disorder of the skin thatcan cause considerable discomfort. It is a disease for which there is nocure and affects people of all ages. Psoriasis affects approximately twopercent of the populations of European and North America. Althoughindividuals with mild psoriasis can often control their disease withtopical agents, more than one million patients worldwide requireultraviolet or systemic immunosuppressive therapy. Unfortunately, theinconvenience and risks of ultraviolet radiation and the toxicities ofmany therapies limit their long-term use. Moreover, patients usuallyhave recurrence of psoriasis, and in some cases rebound, shortly afterstopping immunosuppressive therapy.

Differentiation is a progressive and dynamic process, beginning withpluripotent stem cells and ending with terminally differentiated cells.Pluripotent stem cells that can regenerate without commitment to alineage express a set of differentiation markers that are lost whencommitment to a cell lineage is made. Progenitor cells express a set ofdifferentiation markers that may or may not continue to be expressed asthe cells progress down the cell lineage pathway toward maturation.Differentiation markers that are expressed exclusively by mature cellsare usually functional properties such as cell products, enzymes toproduce cell products, and receptors. The stage of a cell population'sdifferentiation is monitored by identification of markers present in thecell population.

There is evidence to suggest that factors that stimulate specific celltypes down a pathway towards terminal differentiation ordedifferentiation affect the entire cell population originating from acommon precursor or stem cell.

A multimeric cytokine receptor of the present invention can be usefulfor stimulating proliferation, activation, differentiation and/orinduction or inhibition of specialized cell function of cells of theinvolved homeostasis of the hematopoiesis and immune function. Inparticular, multimeric cytokine receptors as described herein are usefulfor stimulating proliferation, activation, differentiation, induction orinhibition of specialized cell functions of cells of the hematopoieticlineages, including, but not limited to, T cells, B cells,monocytes/macrophages, NK cells, neutrophils, endothelial cells,fibroblasts, eosinophils, chondrocytes, mast cells, langerhan cells,monocytes, and macrophages, as well as epithelial cells. Epithelialcells include, for example, ameloblasts, chief cells, chromatophores,enterochramaffin cells, enterochromaffin-like cells, goblet cells,granulosa cells, keratinocytes, dendritic cells, labyrinth supportingcells, melanocytes, merkel cells, paneth cells, parietal cells, sertolicells, and the like.

The present invention also provides a method for reducing hematopoieticcells and hematopoietic cell progenitors of a mammal. The methodincludes culturing bone marrow or peripheral blood cells with acomposition comprising an effective amount of a soluble multimericcytokine receptor to produce a decrease in the number of lymphoid cellsin the bone marrow or peripheral blood cells as compared to bone marrowor peripheral blood cells cultured in the absence of the multimericcytokine receptor. The hematopoietic cells and hematopoietic cellprogenitors can be lymphoid cells, such as monocytic cells, macrophages,or T cells.

The present invention also provides a method of inhibiting an immuneresponse in a mammal exposed to an antigen or pathogen. The methodincludes (a) determining directly or indirectly the level of antigen orpathogen present in the mammal; (b) administering a compositioncomprising a soluble multimeric cytokine receptor in an acceptablepharmaceutical vehicle; (c) determining directly or indirectly the levelof antigen or pathogen in the mammal; and (d) comparing the level of theantigen or pathogen in step (a) to the antigen or pathogen level in step(c), wherein a change in the level is indicative of inhibiting an immuneresponse. The method may further include (e) re-administering acomposition comprising a multimeric cytokine receptor in an acceptablepharmaceutical vehicle; (f) determining directly or indirectly the levelof antigen or pathogen in the mammal; and (g) comparing the number ofthe antigen or pathogen level in step (a) to the antigen level in step(f), wherein a change in the level is indicative of inhibiting an immuneresponse.

Alternatively, the method can include (a) determining a level of anantigen- or pathogen-specific antibody; (b) administering a compositioncomprising a soluble multimeric cytokine receptor in an acceptablepharmaceutical vehicle; (c) determining a post administration level ofantigen- or pathogen-specific antibody; (d) comparing the level ofantibody in step (a) to the level of antibody in step (c), wherein adecrease in antibody level is indicative of inhibiting an immuneresponse.

Zcytor17lig was isolated from tissue known to have importantimmunological function and which contain cells that play a role in theimmune system. Zcytor17lig is expressed in CD3+ selected, activatedperipheral blood cells, and it has been shown that zcytor17ligexpression increases after T cell activation. Moreover, results ofexperiments described in the Examples section herein suggest that amultimeric or heterodimeric cytokine receptor of the present inventioncan have an effect on the growth/expansion of monocytes/macrophages,T-cells, B-cells, NK cells and/or differentiated state ofmonocytes/macrophages, T-cells, B-cells, NK cells or these cells'progenitors. Factors that both stimulate proliferation of hematopoieticprogenitors and activate mature cells are generally known, however,proliferation and activation can also require additional growth factors.For example, it has been shown that IL-7 and Steel Factor (c-kit ligand)were required for colony formation of NK progenitors. IL-15 plus IL-2 incombination with IL-7 and Steel Factor was more effective (Mrózek etal., Blood 87:2632-2640, 1996). However, unidentified cytokines may benecessary for proliferation of specific subsets of NK cells and/or NKprogenitors (Robertson et. al., Blood 76:2451-2438, 1990). Similarly,zcytor17lig may act alone or in concert or synergy with other cytokinesto enhance growth, proliferation expansion and modification ofdifferentiation of monocytes/macrophages, T-cells, B-cells or NK cells.

Assays measuring differentiation include, for example, measuring cellmarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284 (1991); Francis, Differentiation 57:63-75 (1994); and Raes,Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171 (1989)).Alternatively, zcytor17lig polypeptide itself can serve as an additionalcell-surface or secreted marker associated with stage-specificexpression of a tissue. As such, direct measurement of zcytor17ligpolypeptide, or its loss of expression in a tissue as it differentiates,can serve as a marker for differentiation of tissues.

Similarly, direct measurement of zcytor17lig polypeptide, or its loss ofexpression in a tissue can be determined in a tissue or in cells as theyundergo tumor progression. Increases in invasiveness and motility ofcells, or the gain or loss of expression of zcytor17lig in apre-cancerous or cancerous condition, in comparison to normal tissue,can serve as a diagnostic for transformation, invasion and metastasis intumor progression. As such, knowledge of a tumor's stage of progressionor metastasis will aid the physician in choosing the most propertherapy, or aggressiveness of treatment, for a given individual cancerpatient. Methods of measuring gain and loss of expression (of eithermRNA or protein) are well known in the art and described herein and canbe applied to zcytor17lig expression. For example, appearance ordisappearance of polypeptides that regulate cell motility can be used toaid diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter,B. R., Cancer and Metast. Rev. 17:449-458, 1999). As an effector of cellmotility, zcytor17lig gain or loss of expression may serve as adiagnostic for lymphoid, B-cell, epithelial, hematopoietic and othercancers.

Moreover, the activity and effect of zcytor17lig on tumor progressionand metastasis can be measured in vivo. Several syngeneic mouse modelshave been developed to study the influence of polypeptides, compounds orother treatments on tumor progression. In these models, tumor cellspassaged in culture are implanted into mice of the same strain as thetumor donor. The cells will develop into tumors having similarcharacteristics in the recipient mice, and metastasis will also occur insome of the models. Appropriate tumor models for our studies include theLewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No.CRL-6323), amongst others. These are both commonly used tumor lines,syngeneic to the C57BL6/J mouse, that are readily cultured andmanipulated in vitro. Tumors resulting from implantation of either ofthese cell lines are capable of metastasis to the lung in C57BL6/J mice.The Lewis lung carcinoma model has recently been used in mice toidentify an inhibitor of angiogenesis (O'Reilly M S, et al. Cell 79:315-328,1994). C57BL6/J mice are treated with an experimental agenteither through daily injection of recombinant protein, agonist orantagonist or a one time injection of recombinant adenovirus. Three daysfollowing this treatment, 10⁵ to 10⁶ cells are implanted under thedorsal skin. Alternatively, the cells themselves may be infected withrecombinant adenovirus, such as one expressing zcytor17lig, beforeimplantation so that the protein is synthesized at the tumor site orintracellularly, rather than systemically. The mice normally developvisible tumors within 5 days. The tumors are allowed to grow for aperiod of up to 3 weeks, during which time they may reach a size of1500-1800 mm³ in the control treated group. Tumor size and body weightare carefully monitored throughout the experiment. At the time ofsacrifice, the tumor is removed and weighed along with the lungs and theliver. The lung weight has been shown to correlate well with metastatictumor burden. As an additional measure, lung surface metastases arecounted. The resected tumor, lungs and liver are prepared forhistopathological examination, immunohistochemistry, and in situhybridization, using methods known in the art and described herein. Theinfluence of the expressed polypeptide in question, e.g., zcytor17lig,on the ability of the tumor to recruit vasculature and undergometastasis can thus be assessed. In addition, aside from usingadenovirus, the implanted cells can be transiently transfected withzcytor17lig. Use of stable zcytor17lig transfectants as well as use ofinduceable promoters to activate zcytor17lig expression in vivo areknown in the art and can be used in this system to assess zcytor17liginduction of metastasis. Moreover, purified zcytor17lig or zcytor17ligconditioned media can be directly injected in to this mouse model, andhence be used in this system. For general reference see, O'Reilly M S,et al. Cell 79:315-328, 1994; and Rusciano D, et al. Murine Models ofLiver Metastasis. Invasion Metastasis 14:349-361, 1995.

A soluble multimeric cytokine receptor of the present invention orantibodies thereto may be useful in treating tumorgenesis, and thereforewould be useful in the treatment of cancer. Zcytor17lig is expressed inactivated T-cells, monocytes and macrophages, and is linked to a regionof the human chromosome wherein translocations are common in leukemias.Moreover, the zcytor17lig is shown to act through a cytokine receptor,zcytor17 multimeric cytokine receptor, which is also expressed inactivated T-cells, monocytes and macrophages. Over stimulation ofactivated T-cells, monocytes and macrophages by zcytor17lig could resultin a human disease state such as an immune cell cancer. As such,identifying zcytor17lig expression, polypeptides (e.g., byanti-zcytor17lig antibodies, zcytor17 soluble multimeric cytokinereceptors (e.g., zcytor17 receptor, heterodimers (e.g.,zcytor17/OSMRbeta, zcytor17/WSX-1), multimers (e.g.,zcytor17/OSMRbeta/WSX-1)), or other zcytor17lig binding partners) canserve as a diagnostic, and can serve as antagonists of zcytor17ligproliferative activity. The ligand could be administered in combinationwith other agents already in use including both conventionalchemotherapeutic agents as well as immune modulators such as interferonalpha. Alpha/beta interferons have been shown to be effective intreating some leukemias and animal disease models, and the growthinhibitory effects of interferon-alpha and zcytor17lig may be additive.

NK cells are thought to play a major role in elimination of metastatictumor cells and patients with both metastases and solid tumors havedecreased levels of NK cell activity (Whiteside et. al., Curr. Top.Microbiol. Immunol. 230:221-244, 1998). An agent that stimulates NKcells would be useful in the elimination of tumors.

The present invention provides a method of reducing proliferation of aneoplastic monocytes/macrophages comprising administering to a mammalwith a monocyte/macrophage neoplasm an amount of a composition includinga soluble multimeric cytokine receptor or antibody thereto sufficient toreduce proliferation of the neoplastic monocytes/macrophages.

The present invention provides a method for inhibiting activation ordifferentiation of monocytes/macrophages. Monocytes are incompletelydifferentiated cells that migrate to various tissues where they matureand become macrophages. Macrophages play a central role in the immuneresponse by presenting antigen to lymphocytes and play a supportive roleas accessory cells to lymphocytes by secreting numerous cytokines.Macrophages can internalize extracellular molecules and upon activationhave an increased ability to kill intracellular microorganisms and tumorcells. Activated macrophages are also involved in stimulating acute orlocal inflammation.

In another aspect, the present invention provides a method of reducingproliferation of a neoplastic B or T-cells comprising administering to amammal with a B or T cell neoplasm an amount of a composition includinga soluble multimeric cytokine receptor sufficient to reducingproliferation of the neoplastic monocytes/macrophages. Furthermore, thezcytor17lig antagonist can be a ligand/toxin fusion protein.

A zcytor17 multimeric cytokine receptor-saporin fusion toxin may beemployed against a similar set of leukemias and lymphomas, extending therange of leukemias that can be treated with a cytokine antagonsist. Forexample, such leukemias can be those that over-express zcytor17receptors (e.g., zcytor17 receptor, heterodimers (e.g.,zcytor17/OSMRbeta, zcytor17/WSX-1), multimers (e.g.,zcytor17/OSMRbeta/WSX)). Fusion toxin mediated activation of thezcytor17 receptor, zcytor17 receptor heterodimers or multimers (e.g.,zcytor17/OSMRbeta, zcytor17/WSX-1or zcytor17/WSX-1/OSMR) provides twoindependent means to inhibit the growth of the target cells, the firstbeing identical to the effects seen by the ligand alone, and the seconddue to delivery of the toxin through receptor internalization. Thelymphoid and monocyte restricted expression pattern of the zcytor17receptor suggests that the ligand-saporin conjugate can be tolerated bypatients.

The tissue distribution of receptors for a given cytokine offers astrong indication of the potential sites of action of that cytokine.Expression of zcytor17 was seen in monocytes and B-cells, with adramatic increase of expression upon activation for CD3+, CD4+, and CD8+T-cells. In addition, two monocytic cell lines, THP-1 (Tsuchiya et al.,Int. J. Cancer 26:171-176, 1980) and U937 (Sundstrom et al., Int. J.Cancer 17:565-577, 1976), were also positive for zcytor17 expression.

Northern analysis of WSX-1 receptor revealed transcripts in all tissuesexamined, with increased levels of expression in human spleen, thymus,lymph node, bone marrow, and peripheral blood leukocytes. Also,expression levels of WSX-1 increased upon activation of T-cells.

Expression of OSMR is reported to be very broad (Mosley et al, JBC271:32635-32643, 1996). This distribution of zcytor17, WSX-1, andOSMRbeta receptors supports a role for zcytor17lig in immune responses,especially expansion of T-cells upon activation or a role in themonocyte/macrophage arm of the immune system.

Thus, particular embodiments of the present invention are directedtoward use of a soluble multimeric cytokine receptor, for instancezcytor17/WSX-1/OSMR, and zcytor17/OSMR heterodimers, as antagonists ininflammatory and immune diseases or conditions such as pancreatitis,type I diabetes (IDDM), pancreatic cancer, pancreatitis, Graves Disease,inflammatory bowel disease (IBD), Crohn's Disease, colon and intestinalcancer, diverticulosis, autoimmune disease, sepsis, organ or bone marrowtransplant; inflammation due to trauma, surgery or infection;amyloidosis; splenomegaly; graft versus host disease; and whereinhibition of inflammation, immune suppression, reduction ofproliferation of hematopoietic, immune, inflammatory or lymphoid cells,macrophages, T-cells (including Th1 and Th2 cells, CD4+ and CD8+ cells),suppression of immune response to a pathogen or antigen. Moreover thepresence of zcytor17 expression in activated immune cells such asactivated CD4+ and CD19+ cells showed that zcytor17 receptor may beinvolved in the body's immune defensive reactions against foreigninvaders: such as microorganisms and cell debris, and could play a rolein immune responses during inflammation and cancer formation. As such,antibodies and binding partners of the present invention that areagonistic or antagonistic to zcytor17 receptor function, such as asoluble zcytor17 multimeric cytokine receptor, can be used to modifyimmune response and inflammation.

The zcytor17lig structure and tissue expression suggests a role in earlyhematopoietic or thymocyte development and immune response regulation orinflammation. These processes involve stimulation of cell proliferationand differentiation in response to the binding of one or more cytokinesto their cognate receptors. In view of the tissue distribution observedfor this zcytor17lig, agonists (including the natural receptor(s)) andantagonists have enormous potential in both in vitro and in vivoapplications. Compounds identified as zcytor17lig agonists are usefulfor stimulating proliferation and development of target cells in vitroand in vivo. For example, agonist compounds, zcytor17lig, oranti-zcytor17lig antibodies, are useful as components of defined cellculture media, and may be used alone or in combination with othercytokines and hormones to replace serum that is commonly used in cellculture. Agonists are thus useful in specifically promoting the growthand/or development or activation of monocytes, T-cells, B-cells, andother cells of the lymphoid and myeloid lineages, and hematopoieticcells in culture.

The molecules of the present invention have particular use in themonocyte/macrophage arm of the immune system. Methods are known that canassess such activity. For example, interferon gamma (IFNγ) is a potentactivator of mononuclear phagocytes. For example, an increase inexpression of zcytor17 upon activation of THP-1 cells (ATCC No. TIB-202)with interferon gamma could suggest that this receptor is involved inmonocyte activation. Monocytes are incompletely differentiated cellsthat migrate to various tissues where they mature and becomemacrophages. Macrophages play a central role in the immune response bypresenting antigen to lymphocytes and play a supportive role asaccessory cells to lymphocytes by secreting numerous cytokines.Macrophages can internalize extracellular molecules and upon activationhave an increased ability to kill intracellular microorganisms and tumorcells. Activated macrophages are also involved in stimulating acute orlocal inflammation. Moreover, monocyte-macrophage function has beenshown to be abnormal in a variety of diseased states. For example see,Johnston, R B, New Eng. J. Med. 318:747-752, 1998.

One of skill in the art would recognize that agonists of zcytor17multimeric cytokine receptor, such as zcytor17lig, are useful. Forexample, depressed migration of monocytes has been reported inpopulations with a predisposition to infection, such as newborn infants,patients receiving corticosteroid or other immunosuppressive therapy,and patients with diabetes mellitus, burns, or AIDS. Agonists forzcytor17 multimeric cytokine receptor, such as zcytor17lig, could resultin an increase in the ability of monocytes to migrate and possiblyprevent infection in these populations. There is also a profound defectof phagocytic killing by mononuclear phagocytes from patients withchronic granulomatous disease. This results in the formation ofsubcutaneous abscesses, as well as abscesses in the liver, lungs,spleen, and lymph nodes. An agonist of zcytor17 multimeric cytokinereceptor, such as zcytor17lig, could correct or improve this phagocyticdefect. In addition, defective monocyte cytotoxicity has been reportedin patients with cancer and Wiskott-Aldrich syndrome (eczema,thrombocytopenia, and recurrent infections). Activation of monocytes byagonists of zcytor17 multimeric cytokine receptor, such as zcytor17lig,could aid in treatment of these conditions. The monocyte-macrophagesystem is prominently involved in several lipid-storage diseases(sphingolipidoses) such as Gaucher's disease. Resistance to infectioncan be impaired because of a defect in macrophage function, which couldbe treated by agonists to zcytor17 multimeric cytokine receptor such aszcytor17lig.

Moreover, one of skill in the art would recognize that antagonists of azcytor17 multimeric cytokine receptor are useful. For example, inatherosclerotic lesions, one of the first abnormalities is localizationof monocyte/macrophages to endothelial cells. These lesions could beprevented by use of antagonists to zcytor17lig. Zcytor17 solublemultimeric cytokine receptors, such as, for instance, heterodimers andtrimers, can also be used as antagonists to the zcytor17lig. Moreover,monoblastic leukemia is associated with a variety of clinicalabnormalities that reflect the release of the biologic products of themacrophage, examples include high levels of lysozyme in the serum andurine and high fevers. Moreover, such leukemias exhibit an abnormalincrease of monocytic cells. These effects could possibly be preventedby antagonists to zcytor17lig, such as described herein.

Using methods known in the art, and disclosed herein, one of skill couldreadily assess the activity of a zcytor17 multimeric cytokine receptorin the disease states disclosed herein, inflammation, cancer, orinfection as well as other disease states involving monocytic cells. Inaddition, as zcytor17lig is expressed in a T-cell, macrophage andmonocyte-specific manner, and these diseases involve abnormalities inmonocytic cells, such as cell proliferation, function, localization, andactivation, the polynucleotides, polypeptides, and antibodies of thepresent invention can be used to as diagnostics to detect such monocyticcell abnormalities, and indicate the presence of disease. Such methodsinvolve taking a biological sample from a patient, such as blood,saliva, or biopsy, and comparing it to a normal control sample.Histological, cytological, flow cytometric, biochemical and othermethods can be used to determine the relative levels or localization ofzcytor17lig, or cells expressing zcytor17lig, i.e., monocytes, in thepatient sample compared to the normal control. A change in the level(increase or decrease) of zcytor17lig expression, or a change in numberor localization of monocytes (e.g., increase or infiltration ofmonocytic cells in tissues where they are not normally present) comparedto a control would be indicative of disease. Such diagnostic methods canalso include using radiometric, fluorescent, and colorimetric tagsattached to polynucleotides, polypeptides or antibodies of the presentinvention. Such methods are well known in the art and disclosed herein.

Amino acid sequences having zcytor17lig activity can be used to modulatethe immune system by binding soluble zcytor17 multimeric cytokinereceptor, and thus, preventing the binding of zcytor17lig withendogenous zcytor17lig receptor. Zcytor17lig antagonists, such as azcytor17 multimeric cytokine receptor, can also be used to modulate theimmune system by inhibiting the binding of Zcytor17 ligand with theendogenous zcytor17lig receptor. Accordingly, the present inventionincludes the use of a multimeric cytokine receptor that can be also usedto treat a subject which produces an excess of either zcytor17lig orZcytor17 comprising receptor(s). Suitable subjects include mammals, suchas humans or veterinary animals.

Zcytor17lig has been shown to be expressed in activated mononuclearcells, and may be involved in regulating inflammation. As such,polypeptides of the present invention can be assayed and used for theirability to modify inflammation, or can be used as a marker forinflammation. Methods to determine proinflammatory and antiinflammatoryqualities of zcytor17lig are known in the art and discussed herein.Moreover, it may be involved in up-regulating the production of acutephase reactants, such as serum amyloid A (SAA), α1-antichymotrypsin, andhaptoglobin, and that expression of zcytor17 receptor ligand may beincreased upon injection of lipopolysaccharide (LPS) in vivo that areinvolved in inflammatory response (Dumoutier, L. et al., Proc. Nat'l.Acad. Sci. 97:10144-10149 (2000)). Production of acute phase proteins,such as SAA, is considered a short-term survival mechanism whereinflammation is beneficial; however, maintenance of acute phase proteinsfor longer periods contributes to chronic inflammation and can beharmful to human health. For review, see Uhlar, C M and Whitehead, A S,Eur. J. Biochem. 265:501-523 (1999); and Baumann H. and Gauldie, J.Immunology Today 15:74-80 (1994). Moreover, the acute phase protein SAAis implicated in the pathogenesis of several chronic inflammatorydiseases, is implicated in atherosclerosis and rheumatoid arthritis, andis the precursor to the amyloid A protein deposited in amyloidosis(Uhlar, C M and Whitehead, supra.). Thus, where a ligand such aszcytor17lig that acts as a pro-inflammatory molecule and inducesproduction of SAA, antagonists would be useful in treating inflammatorydisease and other diseases associated with acute phase response proteinsinduced by the ligand. Such antagonists are provided by the presentinvention. For example, a method of reducing inflammation comprisesadministering to a mammal with or at risk of developing inflammation anamount of a composition of a soluble multimeric cytokine receptor thatis sufficient to reduce inflammation. Moreover, a method of suppressingan inflammatory response in a mammal with inflammation can comprise: (1)determining a level of serum amyloid A protein; (2) administering acomposition comprising a soluble multimeric cytokine receptorpolypeptide as described herein in an acceptable pharmaceutical vehicle;(3) determining a post administration level of serum amyloid A protein;(4) comparing the level of serum amyloid A protein in step (1) to thelevel of serum amyloid A protein in step (3), wherein a lack of increaseor a decrease in serum amyloid A protein level is indicative ofsuppressing an inflammatory response.

Like zcytor17lig, analysis of the tissue distribution of the mRNAcorresponding it's zcytor17 receptor cDNA showed that mRNA level washighest in monocytes and prostate cells, and is elevated in activatedmonocytes, and activated CD4+, activated CD8+, and activated CD3+ cells.Hence, zcytor17 receptor is also implicated in inducing inflammatory andimmune response. Thus, particular embodiments of the present inventionare directed toward use of zcytor17lig-antibodies, and zcytor17lig, aswell as soluble zcytor17 receptor heterodimers as antagonists ininflammatory and immune diseases or conditions such as pancreatitis,type I diabetes (IDDM), pancreatic cancer, pancreatitis, Graves Disease,inflammatory bowel disease (IBD), Crohn's Disease, colon and intestinalcancer, diverticulosis, autoimmune disease, sepsis, organ or bone marrowtransplant; inflammation due to trauma, sugery or infection;amyloidosis; splenomegaly; graft versus host disease; and whereinhibition of inflammation, immune suppression, reduction ofproliferation of hematopoietic, immune, inflammatory or lymphoid cells,macrophages, T-cells (including Th1 and Th2 cells, CD4+ and CD8+ cells),suppression of immune response to a pathogen or antigen. Moreover thepresence of zcytor17 receptor and zcytor17lig expression in activatedimmune cells such as activated CD3+, monocytes, CD4+ and CD19+ cellsshowed that zcytor17 receptor may be involved in the body's immunedefensive reactions against foreign invaders: such as microorganisms andcell debris, and could play a role in immune responses duringinflammation and cancer formation. As such, zcytor17lig andzcytor17lig-antibodies of the present invention that are agonistic orantagonistic to zcytor17 receptor function, can be used to modify immuneresponse and inflammation.

Moreover, zcytor17lig polypeptides that bind zcytor17 multimericcytokine receptors and antibodies thereto are useful to:

Antagonize or block signaling via a zcytor17 multimeric cytokinereceptor in the treatment of acute inflammation, inflammation as aresult of trauma, tissue injury, surgery, sepsis or infection, andchronic inflammatory diseases such as asthma, inflammatory bowel disease(IBD), chronic colitis, splenomegaly, rheumatoid arthritis, recurrentacute inflammatory episodes (e.g., tuberculosis), and treatment ofamyloidosis, and atherosclerosis, Castleman's Disease, asthma, and otherdiseases associated with the induction of acute-phase response.

Antagonize or block signaling via the zcytor17 multimeric cytokinereceptor in the treatment of autoimmune diseases such as IDDM, multiplesclerosis (MS), systemic Lupus erythematosus (SLE), myasthenia gravis,rheumatoid arthritis, and IBD to prevent or inhibit signaling in immunecells (e.g. lymphocytes, monocytes, leukocytes) via zcytor17 receptor(Hughes C et al., J. Immunol 153: 3319-3325 (1994)). Asthma, allergy andother atopic disease may be treated with an MAb against, for example,soluble zcytor17 multimeric cytokine receptors or zcytor17/CRF2-4heterodimers, to inhibit the immune response or to deplete offendingcells. Blocking or inhibiting signaling via zcytor17 multimeric cytokinereceptor, using the polypeptides and antibodies of the presentinvention, may also benefit diseases of the pancreas, kidney, pituitaryand neuronal cells. IDDM, NIDDM, pancreatitis, and pancreatic carcinomamay benefit. Zcytor17 multimeric cytokine receptor may serve as a targetfor MAb therapy of cancer where an antagonizing MAb inhibits cancergrowth and targets immune-mediated killing. (Holliger P, and Hoogenboom,H Nature Biotech. 16: 1015-1016 (1998)). Mabs to soluble zcytor17receptor monomers, homodimers, heterodimers and multimers may also beuseful to treat nephropathies such as glomerulosclerosis, membranousneuropathy, amyloidosis (which also affects the kidney among othertissues), renal arteriosclerosis, glomerulonephritis of various origins,fibroproliferative diseases of the kidney, as well as kidney dysfunctionassociated with SLE, IDDM, type TI diabetes (IDDM), renal tumors andother diseases.

Agonize or initiate signaling via the zcytor17 multimeric cytokinereceptor in the treatment of autoimmune diseases such as IDDM, MS, SLE,myasthenia gravis, rheumatoid arthritis, and IBD. Zcytor17lig may signallymphocytes or other immune cells to differentiate, alter proliferation,or change production of cytokines or cell surface proteins thatameliorate autoimmunity. Specifically, modulation of a T-helper cellresponse to an alternate pattern of cytokine secretion may deviate anautoimmune response to ameliorate disease (Smith J A et al., J. Immunol.160:4841-4849 (1998)). Similarly, zcytor17lig may be used to signal,deplete and deviate immune cells involved in asthma, allergy and atopoicdisease. Signaling via zcytor17 multimeric cytokine receptor may alsobenefit diseases of the pancreas, kidney, pituitary and neuronal cells.IDDM, NIDDM, pancreatitis, and pancreatic carcinoma may benefit.Zcytor17 multimeric cytokine receptor may serve as a target for MAbtherapy of pancreatic cancer where a signaling MAb inhibits cancergrowth and targets immune-mediated killing (Tutt, A L et al., J Immunol.161: 3175-3185 (1998)). Similarly T-cell specific leukemias, lymphomas,plasma cell dyscrasia (e.g., multiple myeloma), and carcinoma may betreated with monoclonal antibodies (e.g., neutralizing antibody) tozcytor17-comprising soluble receptors of the present invention.

Soluble zcytor17 multimeric cytokine receptors as described herein canbe used to neutralize/block zcytor17 receptor ligand activity in thetreatment of autoimmune disease, atopic disease, NIDDM, pancreatitis andkidney dysfunction as described above. A soluble form of zcytor17multimeric cytokine receptor may be used to promote an antibody responsemediated by T cells and/or to promote the production of IL-4 or othercytokines by lymphocytes or other immune cells.

A soluble zcytor17 multimeric cytokine receptor may be useful asantagonists of zcytor17lig. Such antagonistic effects can be achieved bydirect neutralization or binding of its natural ligand. In addition toantagonistic uses, the soluble receptors can bind zcytor17lig and act ascarrier or vehicle proteins, in order to transport zcytor17lig todifferent tissues, organs, and cells within the body. As such, thesoluble receptors can be fused or coupled to molecules, polypeptides orchemical moieties that direct the soluble-receptor-ligand complex to aspecific site, such as a tissue, specific immune cell, monocytes, ortumor. For example, in acute infection or some cancers, benefit mayresult from induction of inflammation and local acute phase responseproteins. Thus, the soluble receptors described herein or antibodiesthereto can be used to specifically direct the action of apro-inflammatory zcytor17lig ligand. See, Cosman, D. Cytokine 5: 95-106(1993); and Fernandez-Botran, R. Exp. Opin. Invest. Drugs 9:497-513(2000).

Moreover, the soluble zcytor17 multimeric cytokine receptors can be usedto stabilize the zcytor17lig, to increase the bioavailability,therapeutic longevity, and/or efficacy of the ligand by stabilizing theligand from degradation or clearance, or by targeting the ligand to asite of action within the body. For example, the naturally occurringIL-6/soluble IL-6R complex stabilizes IL-6 and can signal through thegp130 receptor. See, Cosman, D. supra., and Fernandez-Botran, R. supra.Moreover, Zcytor17 may be combined with a cognate ligand such as itsligand to comprise a ligand/soluble receptor complex. Such complexes maybe used to stimulate responses from cells presenting a companionreceptor subunit. The cell specificity of zcytor17 multimeric cytokinereceptor/zcytor17lig complexes may differ from that seen for the ligandadministered alone. Furthermore the complexes may have distinctpharmacokinetic properties such as affecting half-life, dose/responseand organ or tissue specificity. Zcytor17 multimeric cytokinereceptor/ligand complexes thus may have agonist activity to enhance animmune response or stimulate mesangial cells or to stimulate hepaticcells. Alternatively, only tissues expressing a signaling subunit theheterodimerizes with the complex may be affected analogous to theresponse to IL6/IL6R complexes (Hirota H. et al., Proc. Nat'l. Acad.Sci. 92:4862-4866 (1995); and Hirano, T. in Thomason, A. (Ed.) “TheCytokine Handbook”, 3^(rd) Ed., p. 208-209). Soluble receptor/cytokinecomplexes for IL12 and CNTF display similar activities.

Zcytor17lig may also be used within diagnostic systems for the detectionof circulating levels of ligand, and in the detection of acute phaseinflammatory response. Within a related embodiment, antibodies or otheragents that specifically bind to zcytor17lig can be used to detectcirculating zcytor17lig polypeptides; conversely, zcytor17lig itself canbe used to detect circulating or locally-acting receptor polypeptides.Elevated or depressed levels of ligand or receptor polypeptides may beindicative of pathological conditions, including inflammation or cancer.Moreover, detection of acute phase proteins or molecules such aszcytor17lig can be indicative of a chronic inflammatory condition incertain disease states (e.g., rheumatoid arthritis). Detection of suchconditions serves to aid in disease diagnosis as well as help aphysician in choosing proper therapy.

Polynucleotides encoding a zcytor17 multimeric cytokine receptor areuseful within gene therapy applications where it is desired to increaseor inhibit zcytor17lig activity. If a mammal has a mutated or absentzcytor17 gene, the zcytor17 gene of the present invention can beintroduced into the cells of the mammal. In one embodiment, a geneencoding a zcytor17 multimeric cytokine receptor is introduced in vivoin a viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30 (1991)); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30(1992); and a defective adeno-associated virus vector (Samulski et al.,J. Virol. 61:3096-101 (1987); and Samulski et al., J. Virol. 63:3822-8(1989)).

A zcytor17 gene of the present invention can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153 (1983); Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120 (1988); Temin et al., U.S. Pat. No. 5,124,263;International Patent Publication No. WO 95/07358, published Mar. 16,1995 by Dougherty et al.; and Kuo et al., Blood 82:845 (1993).Alternatively, the vector can be introduced by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7 (1987); Mackey et al., Proc. Natl.Acad. Sci. USA 85:8027-31 (1988)). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. More particularly, directingtransfection to particular cells represents one area of benefit. Forinstance, directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, suchas the immune system, pancreas, liver, kidney, and brain. Lipids may bechemically coupled to other molecules for the purpose of targeting.Targeted peptides (e.g., hormones or neurotransmitters), proteins suchas antibodies, or non-peptide molecules can be coupled to liposomeschemically.

It is possible to remove the target cells from the body; to introducethe vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7 (1992); and Wu et al., J. Biol. Chem. 263:14621-4 (1988).

Antisense methodology can be used to inhibit zcytor17 multimericcytokine receptor gene transcription, such as to inhibit cellproliferation in vivo. Polynucleotides that are complementary to asegment of a zcytor17-encoding polynucleotide (e.g., a polynucleotide asset forth in SEQ ID NO:110. SEQ ID NO:108, or SEQ ID NO:4) are designedto bind to zcytor17lig-encoding mRNA and to inhibit translation of suchmRNA. Such antisense polynucleotides are used to inhibit expression ofzcytor17lig polypeptide-encoding genes in cell culture or in a subject.

Mice engineered to express the zcytor17lig gene, referred to as“transgenic mice,” and mice that exhibit a complete absence ofzcytor17lig gene function, referred to as “knockout mice,” may also begenerated (Snouwaert et al., Science 257:1083 (1992); Lowell et al.,Nature 366:740-42 (1993); Capecchi, M. R., Science 244: 1288-1292(1989); Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499 (1986)). Forexample, transgenic mice that over-express zcytor17lig, eitherubiquitously or under a tissue-specific or tissue-restricted promotercan be used to ask whether over-expression causes a phenotype. Forexample, over-expression of a wild-type zcytor17lig polypeptide,polypeptide fragment or a mutant thereof may alter normal cellularprocesses, resulting in a phenotype that identifies a tissue in whichzcytor17lig expression is functionally relevant and may indicate atherapeutic target for the zcytor17lig, its agonists or antagonists. Forexample, a preferred transgenic mouse to engineer is one thatover-expresses the zcytor17lig (amino acid residues 23-164 of SEQ IDNO:2; or 24-163 of SEQ ID NO:11). Moreover, such over-expression mayresult in a phenotype that shows similarity with human diseases.Similarly, knockout zcytor17lig mice can be used to determine wherezcytor17lig is absolutely required in vivo. The phenotype of knockoutmice is predictive of the in vivo effects of that a zcytor17ligantagonist, such as a soluble zcytor17 multimeric cytokine receptor, mayhave. The human or mouse zcytor17lig cDNA described herein can be usedto generate knockout mice. These mice may be employed to study thezcytor17lig gene and the protein encoded thereby in an in vivo system,and can be used as in vivo models for corresponding human diseases.Moreover, transgenic mice expression of zcytor17lig antisensepolynucleotides or ribozymes directed against zcytor17lig, describedherein, can be used analogously to transgenic mice described above.Studies may be carried out by administration of purified zcytor17ligprotein, as well.

The present invention also provides a composition which includes aneffective amount of a soluble multimeric cytokine receptor comprising apolypeptide comprising amino acid residue 20 to amino acid residue 543of SEQ ID NO:111 and at least a portion of at least one class I cytokinereceptor; and a pharmaceutically acceptable vehicle. The polypeptide maybe comprised of various fragement or portions of the extracellulardomain of SEQ ID NO:111, SEQ ID NO:109, and/or SEQ ID NO:5, such as forinstance, amino acid residue 20 to amino acid residue 227 of SEQ IDNO:111 and amino acid residue 20 to amino acid residue 519 of SEQ IDNO:111. The at least a portion of at least one class I cytokine receptorcan include, for example, a portion of SEQ ID NO:9 and/or a portion ofSEQ ID NO:7, such as, for instance, amino acid residue 28 to amino acidresidue 429 of SEQ ID NO:7, amino acid residue 35 to amino acid residue137 of SEQ ID NO:7, amino acid residue 240 to amino acid residue 342 ofSEQ ID NO:7, amino acid residue 348 to amino acid residue 429 of SEQ IDNO:7, amino acid residue 28 to amino acid residue 739 of SEQ ID NO:7,and/or combinations thereof. The multimeric cytokine receptor mayfurther include an affinity tag as described herein.

The present invention also provides an immune cell inhibitingcomposition which includes an effective amount of a soluble multimericcytokine receptor comprising a polypeptide comprising amino acid residue20 to amino acid residue 227 of SEQ ID NO:111 and at least a portion ofat least one class I cytokine receptor; and a pharmaceuticallyacceptable vehicle, wherein the soluble multimeric cytokine receptorinhibits the proliferation of immune cells.

The present invention also provides an inflammatory cell inhibitingcomposition which includes an effective amount of a soluble multimericcytokine receptor comprising a polypeptide comprising amino acid residue20 to amino acid residue 227 of SEQ ID NO:111 and at least a portion ofat least one class I cytokine receptor; and a pharmaceuticallyacceptable vehicle, wherein the soluble multimeric cytokine receptorinhibits the proliferation of inflammatory cells.

Experimental evidence suggests a role for zcytor17lig in the progressionof diseases that involve the skin or epithelium of internal surfaces,such as, for instance, large intestine, small intestine, pancrease,lung, prostate, uterus, and the like. First, as disclosed herein,zcytor17 receptors, including both OSM receptor beta and zcytor17, areexpressed in several cell types located in epithelial surfaces includingcell lines derived from lung epithelium, lung fibroblast, prostate,colon, breast, liver epithelium, bone and skin epithelium, bonefibroblast, and the like. Moreover, as disclosed herein, examples fromeach of these cell types also responded to zcytor17lig activation of aSTAT reporter construct. In addition, several cell lines responded tozcytor17lig stimulation by producing increased levels of IL-6, IL-8,MCP-1 (a chemotactic factor) as described herein. In whole, these datasuggest a role for zcytor17lig in diseases that involve the epitheliumsuch as, for instance, atopic dermatitis; dermatitis; psoriasis;psoriatic arthritis; eczema; gingivitis; peridontal disease;inflammatory bowel diseases (IBD) (e.g., ulcerative colitis, Crohn'sdisease); reproductive disorders, such as, for instance, cervicaldysplasia, cervical cancer; other skin diseases like cancers: sarcomas;canrcinomas; melanoma, etc. i.e, not just inflammatory diseases, sinceimmune system is involved in activating/curing cancers; diseasesinvolving barrier dysfunction, such as, for instance, graft-versus-hostdisease (GVHD) and irritable bowel syndrome (IBS); and diseases thatinvolve lung epithelium, such as asthma, emphysema, and the like. Inaddition, the release of cytokines IL-6, IL-8, and MCP-1 by cellsexposed to zcytor17lig suggests that zcytor17lig is involved ininflammation. Therefore, regulation of zcytor17lig can be useful in thetreatment of autoimmune, inflammatory, or cancerous diseases associatedwith the tissues that express receptor. These diseases include, forexample, prostatitis, hepatitis, osteoarthritis, and the like.Zcytor17lig may positively or negatively directly or indirectly regulatethese diseases. Therefore, the administration of zcytor17lig can be usedto treat diseases as described herein directly or with molecules thatinhibit zcytor17lig activity including, for example, both monoclonalantibodies to zcytor17lig or monoclonal antibodies to zcytor17, ormonoclonal antibodies that recognize the zcytor17 and OSM receptor betacomplex.

Data also suggests that zcytor17lig may be involved in the regulation ofTH2 T cell mediated diseases. First, zcytor17lig is made by the TH2subset of activated T cells. TH2 cells express more zcytor17lig ascompared to TH1 cells. In addition, at least two lung epithelial celllines (SK-LU-1, A549) were stimulated to increase IL13 receptor alpha-2mRNA in response to zcyto17 ligand stimulation as described herein.There is an association of IL-13 receptor alpha2 chain andtumorigenicity of human breast and pancreatic tumors. This suggests thatzcytor17lig may play a role in regulating tumorigenicity of these typesof cancers, as well as other cancers. Therefore, the administration of azcytor17lig antagonist or direct use of zcytor17lig may be useful intreatment of these types of cancers, benign or malignant and at variousgrades (grades I-IV) and stages (e.g., TNM or AJC staging methods) oftumor development, in mammals, preferably humans.

It is well-known in the art that IL13 is involved in the generation ofactivated TH2 cells and in TH2 mediated diseases, such as asthma, atopicdermatitis, and the like. Zcytor17lig or zcytor17lig antagonists may beuseful in the treatment of diseases that involved TH2 T cells. Thiswould include diseases such as, for instance, atopic dermatitis, asthma,as well as other diseases that are exacerbated by activated TH2 cells.The involvement of zcytor17lig in diseases, such as, for instance,atopic dermatitis, is also supported by the phenotype of the transgenicmice that overexpress zcytor17lig and develop symptoms of atopicdermatitis as described herein.

Despite the preferential expression of zcytor17lig by TH2 cells, thereis still some expression of zcytor17lig in TH1 cells and in CD8+ Tcells. Therefore, zcytor17lig or its antagonists may be useful intreating diseases that involve immune modulation of activated T cellsincluding, for example, viral infection, cancers, graft rejection, andthe like.

Zcytor17lig may also be involved in the development of cancer. There isexpression of the zcytor17 and OSM receptor beta receptors in human bonefibroblast osteosarcomas, human skin fibroblast melanoma, colonepithelial carcinoma, adenocarcinoma, breast epithelial adenocarcinoma,prostate epithelial adenosarcoma, and lung epithelial adenocarcinoma andcarcinoma. Therefore, it may be useful to treat tumors of epithelialorigin with either zcytor17lig, fragments thereof, or zcytor17ligantagonists which include, but are not limited to, carcinoma,adenocarcinoma, and melanoma. Notwithstanding, zcytor17lig or azcytor17lig antagonist may be used to treat a cancer, or reduce one ormore symptoms of a cancer, from a cancer including but not limited to,squamous cell or epidermoid carcinoma, basal cell carcinoma,adenocarcinoma, papillary carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, bronchial adenoma, melanoma, renal cell carcinoma,hepatocellular carcinoma, transitional cell carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, malignant mixed tumor of salivary glandorigin, Wilms' tumor, immature teratoma, teratocarcinoma, and othertumors comprising at least some cells of epithelial origin.

Generally, the dosage of administered zcytor17lig polypeptide (orZcytor16 analog or fusion protein) will vary depending upon such factorsas the patient's age, weight, height, sex, general medical condition andprevious medical history. Typically, it is desirable to provide therecipient with a dosage of zcytor17lig polypeptide which is in the rangeof from about 1 pg/kg to 10 mg/kg (amount of agent/body weight ofpatient), although a lower or higher dosage also may be administered ascircumstances dictate. One skilled in the art can readily determine suchdosages, and adjustments thereto, using methods known in the art.

Administration of a zcytor17 multimeric receptor agonist or antagonistto a subject can be topical, inhalant, intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal,by perfusion through a regional catheter, or by direct intralesionalinjection. When administering therapeutic proteins by injection, theadministration may be by continuous infusion or by single or multipleboluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprising Zcytor17multimeric receptor agonist or antagonist can be prepared and inhaledwith the aid of dry-powder dispersers, liquid aerosol generators, ornebulizers (e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton etal., Adv. Drug Deliv. Rev. 35:235 (1999)). This approach is illustratedby the AERX diabetes management system, which is a hand-held electronicinhaler that delivers aerosolized insulin into the lungs. Studies haveshown that proteins as large as 48,000 kDa have been delivered acrossskin at therapeutic concentrations with the aid of low-frequencyultrasound, which illustrates the feasibility of trascutaneousadministration (Mitragotri et al., Science 269:850 (1995)). Transdermaldelivery using electroporation provides another means to administer amolecule having Zcytor17 multimeric receptor binding activity (Potts etal., Pharm. Biotechnol. 10:213 (1997)).

A pharmaceutical composition comprising a protein, polypeptide, orpeptide having Zcytor17 multimeric receptor binding activity can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the therapeutic proteins are combined in a mixturewith a pharmaceutically acceptable vehicle. A composition is said to bein a “pharmaceutically acceptable vehicle” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable vehicle. Other suitablevehicles are well-known to those in the art. See, for example, Gennaro(ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, molecules having Zcytor17 multimeric receptorbinding activity and a pharmaceutically acceptable vehicle areadministered to a patient in a therapeutically effective amount. Acombination of a protein, polypeptide, or peptide having Zcytor17multimeric receptor binding activity and a pharmaceutically acceptablevehicle is said to be administered in a “therapeutically effectiveamount” or “effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates at least aportion of the inflammatory response.

A pharmaceutical composition comprising Zcytor17lig (or Zcytor17liganalog or fusion protein) can be furnished in liquid form, in anaerosol, or in solid form. Liquid forms, are illustrated by injectablesolutions, aerosols, droplets, topological solutions and oralsuspensions. Exemplary solid forms include capsules, tablets, andcontrolled-release forms. The latter form is illustrated by miniosmoticpumps and implants (Bremer et at., Pharm. Biotechnol. 10:239 (1997);Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems, Ranadeand Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al.,“Protein Delivery with Infusion Pumps,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);Yewey et al., “Delivery of Proteins from a Controlled Release InjectableImplant,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 93-117 (Plenum Press 1997)). Other solid forms includecreams, pastes, other topological applications, and the like.

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et at., Ann. N.Y Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et at., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet at., Japanese Patent 04-244,018; Kato et at., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull. 20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al, Adv. Drug Deliv. Rev.32:99 (1998)).

Polypeptides having Zcytor17 multimeric receptor binding activity can beencapsulated within liposomes using standard techniques of proteinmicroencapsulation (see, for example, Anderson et al., Infect. Immun.31:1099 (1981), Anderson et al., Cancer Res. 50:1853 (1990), and Cohenet al., Biochim. Biophys. Acta 1063:95 (1991), Alving et al.“Preparation and Use of Liposomes in Immunological Studies,” in LiposomeTechnology, 2nd Edition, Vol. III, Gregoriadis (ed.), page 317 (CRCPress 1993), Wassef et at., Meth. Enzymol. 149:124 (1987)). As notedabove, therapeutically useful liposomes may contain a variety ofcomponents. For example, liposomes may comprise lipid derivatives ofpoly(ethylene glycol) (Allen et at., Biochim. Biophys. Acta 1150:9(1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide vehicles for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

The present invention also contemplates chemically modified polypeptideshaving binding Zcytor17 multimeric receptor activity such as zcytor17multimeric receptor heterodimeric or multimeric soluble receptors, andZcytor17 multimeric receptor antagonists, for example anti-zcytor17multimeric receptor antibodies or binding polypeptides, which apolypeptide is linked with a polymer, as discussed above.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises a polypeptide with a Zcytor17multimeric receptor extracellular domain, e.g., zcytor17 multimericreceptor heterodimeric or multimeric soluble receptors, or a Zcytor17multimeric receptor antagonist (e.g., a neutralizing antibody orantibody fragment that binds a Zcytor17 multimeric receptorpolypeptide). Therapeutic polypeptides can be provided in the form of aninjectable solution for single or multiple doses, or as a sterile powderthat will be reconstituted before injection. Alternatively, such a kitcan include a dry-powder disperser, liquid aerosol generator, ornebulizer for administration of a therapeutic polypeptide. Such a kitmay further comprise written information on indications and usage of thepharmaceutical composition. Moreover, such information may include astatement that the Zcytor17 multimeric receptor composition iscontraindicated in patients with known hypersensitivity to Zcytor17multimeric receptor.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (e.g., GenBank aminoacid and nucleotide sequence submissions) cited herein are incorporatedby reference. The foregoing detailed description and examples have beengiven for clarity of understanding only. No unnecessary limitations areto be understood therefrom. The invention is not limited to the exactdetails shown and described, for variations obvious to one skilled inthe art will be included within the invention defined by the claims.

EXAMPLES Example 1 Construction of MPL-Zcytor17 Polypeptide Chimera: MPLExtracellular and TM Domain Fused to the zcytor17 IntracellularSignaling Domain

The 5′ extracellular domain of the murine MPL receptor was isolated froma plasmid containing the murine MPL receptor (PHZ1/MPL plasmid) bydigestion with EcoRI and BamHI generating a 1164 bp fragment. Thedigestion was run on a 1% agarose gel and the fragment was isolatedusing the Qiaquick gel extraction kit (Qiagen) as per manufacturer'sinstructions. The rest of the MPL extracellular domain and transmembranedomain were generated using PCR with primers ZC6,673 (SEQ ID NO:13) andZC29,082 (SEQ ID NO:14). The reaction conditions were as follows: 15cycles at 94° C. for 1 min., 55° C. for 1 min., 72° C. for 2 min.;followed by 72° C. for 7 min.; then a 4° C. soak. The PCR product wasrun on a 1% agarose gel and the approximately 400 bp MPL receptorfragment was isolated using Qiaquick™ gel extraction kit (Qiagen) as permanufacturer's instructions.

The intracellular domain of human zcytor17 was isolated from a plasmidcontaining zcytor17 receptor cDNA (#23/pCAP) using PCR with primersZC29,083 (SEQ ID NO:15) and ZC29,145 (SEQ ID NO:16). The polynucleotidesequence that corresponds to the zcytor17 receptor coding sequence isshown in SEQ ID NO:5. The reaction conditions were as per above. The PCRproduct was run on a 1% agarose gel and the approximately 320 bpzcytor17 fragment isolated using Qiaquick gel extraction kit as permanufacturer's instructions.

Each of the isolated PCR fragments described above were mixed at a 1:1volumetric ratio and used in a PCR reaction using ZC6673 (SEQ ID NO:13)and ZC29145 (SEQ ID NO:16) to create all but the 5′ MPL portion of theMPL-zcytor17 chimera. The reaction conditions were as follows: 15 cyclesat 94° C. for 1 min., 55° C. for 1 min., 72° C. for 2 min.; followed by72° C. for 7 min.; then a 4° C. soak. The entire PCR product was run ona 1% agarose gel and the approximately 700 bp MPL-zcytor17 chimerafragment isolated using Qiaquick gel extraction kit (Qiagen) as permanufacturer's instructions. The MPL-zcytor17 chimera fragment wasdigested with BamHI (BRL) and XbaI (Boerhinger Mannheim) as permanufacturer's instructions. The entire digest was run on a 1% agarosegel and the cleaved MPL-zcytor17 chimera isolated using Qiaquick™ gelextraction kit (Qiagen) as per manufacturer's instructions. Theresultant cleaved MPL-zvytor17 chimera plus 5′ MPL EcoRI/BamHI fragmentdescribed above were inserted into an expression vector to generate thefull MPL-zcytor17 chimeric receptor as described below.

Recipient expression vector pZP-7 was digested with EcoRI (BRL) and Xba1(BRL) as per manufacturer's instructions, and gel purified as describedabove. This vector fragment was combined with the EcoRI and XbaI cleavedMPL-zcytor17 PCR chimera isolated above and the EcoRI and BamHI 5′ MPLfragment isolated above in a ligation reaction. The ligation was runusing T4 Ligase (Epicentre Technologies), at room temperature for 1 houras per manufacturer's instructions. A sample of the ligation waselectroporated into DHIOB ElectroMAX™ electrocompetent E. coli cells (25μF, 200Ω, 1.8V). Transformants were plated on LB+Ampicillin plates andsingle colonies screened by miniprep (Qiagen) and digestion with EcoRIto check for the MPL-zcytor17 chimera. EcoRI digestion of correct clonesyield about a 2 kb fragment. Confirmation of the MPL-zcytor17 chimerasequence was made by sequence analyses. The insert was approximately 3.1kb, and was full-length.

Example 2 MPL-Zcytor17 Chimera Based Proliferation in BAF3 Assay UsingAlamar Blue

A. Construction of BaF3 Cells Expressing MPL-zcytor17 Chimera

BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell line derivedfrom murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), wasmaintained in complete media (RPMI medium (JRH Bioscience Inc., Lenexa,Kans.) supplemented with 10% heat-inactivated fetal calf serum, 1-2ng/ml murine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2mM L-glutaMax-1™ (Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics(GIBCO BRL)). Prior to electroporation, pZP-7/MPL-zcytor17 plasmid DNAwas prepared and purified using a Qiagen Maxi Prep kit (Qiagen) as permanufacturer's instructions. BaF3 cells for electroporation were washedtwice in RPMI media and then resuspended in RPMI media at a cell densityof 10⁷ cells/ml. One ml of resuspended BaF3 cells was mixed with 30 μgof the pZP-7/MPL-zcytor17 plasmid DNA and transferred to separatedisposable electroporation chambers (GIBCO BRL). At room temperaturecells were given 5×0.1msec shocks at 800 volts followed by 5×2 ms shocksat 600 volts delivered by an electroporation apparatus (Cyto-Pulse).Alternatively, cells were electroporated with two serial pulses (800μFAD/300 V; followed by 1180 μFAD/300 V) delivered by a Cell-Porator(GibcoBRL) electroporation apparatus. The electroporated cells weretransferred to 50 ml of complete media and placed in an incubator for15-24 hours (37° C., 5% CO2). Then Geneticin™ (Gibco) selection (1 mg/mlG418) was added to the cells in a T-162 flask to isolate theG418-resistant pool. Pools of the transfected BaF3 cells, hereinaftercalled BaF3/MPL-zcytor17 cells, were assayed for signaling capability asdescribed below.

B. Testing the Signaling Capability of the BaF3/MPL-Zcytor17 Cells Usingan Alamar Blue Proliferation Assay

BaF3/MPL-zcytor17 cells were spun down and washed in the complete media,described above, but without mIL-3 (hereinafter referred to as “mIL-3free media”). The cells were spun and washed 3 times to ensure theremoval of the mIL-3. Cells were then counted in a hemacytometer. Cellswere plated in a 96-well format at 5000 cells per well in a volume of100 μl per well using the mIL-3 free media.

Proliferation of the BaF3/MPL-zcytor17 cells was assessed using murinethrombopoietin (mTPO) diluted with mIL-3 free media to 200 ng/ml, 100ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.1 ng/ml, 1.5 ng/mlconcentrations. One hundred microliters of the diluted mTPO was added tothe BaF3/MPL-zcytor17 cells. The total assay volume was 200 μl. Negativecontrols were run in parallel using mIL-3 free media only, without theaddition of mTPO. The assay plates were incubated at 37° C., 5% CO₂ for3 days at which time Alamar Blue (Accumed, Chicago, Ill.) was added at20 μl/well. Alamar Blue gives a fluorometric readout based on themetabolic activity of cells, and is thus a direct measurement of cellproliferation in comparison to a negative control. Plates were againincubated at 37° C., 5% CO₂ for 24 hours. Plates were read on the Fmax™plate reader (Molecular Devices Sunnyvale, Calif.) using the SoftMax™Pro program, at wavelengths 544 (Excitation) and 590 (Emission), or aWallac Victor 2 plate reader (PerkinElmer Life Sciences, Boston, Mass.).

Results confirmed the signaling capability of the intracellular portionof the zcytor17 receptor, as the thrombopoietin induced proliferation atapproximately 9-13 fold over background at mTPO concentrations of 50ng/ml and greater.

Example 3 Construction of Expression Vector Expressing Full-LengthZcytor17. pZp7pX/Zcytor17

A. Cloning of Full Length Zcytor17 cDNA for Expression:

To obtain a full-length zcytor17 cDNA, 5′ and 3′ PCR products wereisolated and joined using an internal PstI site. The PCR primers weredesigned using the nucleotide sequence SEQ ID NO:4 and include BamHI andXho I restriction sites for cloning purposes.

A 5′ PCR product was generated using a WI-38 cDNA library as a templateand oligonucleotides ZC29,359 (SEQ ID NO:18) and ZC27,899 (SEQ ID NO:19)as primers. WI-38 is an in-house cDNA library generated from a humanembryonic lung cell line (ATCC CRL-2221). This 5′ PCR reaction was runas follows: 30 cycles at 94° C. for 1 minute, 65° C. for 1 minute, 72°C. for 2 minutes, then 72° C. for 7 minutes; 10° C. soak. The PCRreaction used approximately 3 μg of plasmid prepared from the cDNAlibrary, 20 pmoles of each oligonucleotide, and five units of PWO DNApolymerase (Roche). About 90% of the 5′ PCR product was ethanolprecipitated, digested with BamHI and PstI and gel purified on a 1.0%agarose gel. The approximately 600 bp band was excised and used forligation to the cloning vector pUC18 digested with BamHI and PstI. Theresulting transformants were sequenced to confirm the zcytor17 cDNAsequence. For one of these transformants, plasmid DNA was prepared anddigested with BamHI and PstI. The resulting approximately 600 bp bandwas gel purified and used for a ligation below to form a full-lengthcDNA.

A 3′ PCR product was generated using a human testes in-house CDNAlibrary as a template and oligonucleotides ZC27,895 (SEQ ID NO:20) andZC29,122 (SEQ ID NO:21) as primers. This 3′ PCR reaction was run asfollows: 30 cycles at 94° C. for 45 seconds, 65° C. for 45 seconds, 72°C. for 2 minutes, then 72° C. for 7 minutes; 10° C. soak. The entire 3′PCR reaction was gel purified on a 1.0% agarose gel and the major 1500bp band excised. This band was cloned into the PCR Blunt II TOPO vectorusing the Zeroblunt TOPO kit (Invitrogen). The resulting transformantswere sequenced to confirm the zcytor17 cDNA sequence. For one of thesetransformants, plasmid DNA was prepared and digested with PstI and XhoI.The resulting approximately 1500 bp band was gel purified. A three-partligation was performed with the 5′ BamHI to Pst I fragment above, the 3′PstI to XhoI fragment, and the expression vector pZp7pX digested withBamHI and XhoI. This generated a pZp7pX plasmid containing a full-lengthcDNA for zcytor17 (SEQ ID NO:4), designated pZp7p/zcytor17. The fulllength zcytor17 cDNA in pZp7p/zcytor17 has a silent mutation thatchanges the T to G at position 1888 of SEQ ID NO:4 (encoding a Glyresidue at residue 464 of SEQ ID NO:5). As this mutation is silent, thezcytor17 cDNA in pZp7p/zcytor17 encodes the polypeptide as shown in SEQID NO:5. Plasmid pZp7pX is a mammalian expression vector containing anexpression cassette having the CMV promoter, intron A, multiplerestriction sites for insertion of coding sequences, and a human growthhormone terminator. The plasmid also has an E. coli origin ofreplication, a mammalian selectable marker expression unit having anSV40 promoter, enhancer and origin of replication, a puromycinresistance gene and the SV40 terminator.

B. Construction of Expression Vector Expressing Full-Length WSX-1

The entire WSX-1 receptor (SEQ ID NO:9) was isolated from a plasmidcontaining the WSX-1 receptor cDNA (SEQ ID NO:8) (U.S. Pat. No.5,925,735). hWSX-1/pBluescript SK(+) plasmid DNA (Stratagene, La Jolla,Calif.) was digested with EcoRI and XhoI to generate a 1075 bp fragment,and also digested with XhoI and XbaI to generate a 900 bp fragment. Bothdigests were run on a 1% agarose gel and the cleaved WSX-1 fragmentsisolated.

Recipient expression vector pZp7Z was digested with EcoRI and XbaI andgel purified as described above. This vector fragment was combined withthe two cleaved zcytor17 fragments isolated above in a ligation reactionusing T4 Ligase (BRL). The ligation was incubated at room temperatureovernight. A sample of the ligation was electroporated in to DH10BelectroMAX™ electrocompetent E. coli cells (25 μF, 200Ω, 2.3V). Sixcolonies were grown in culture and miniprepped DNA was prepared anddigested to confirm the correct WSX-1 full-length insert of 2.0 kb. Theresulting plasmid is pZPZ7Z/WSX-1.

Example 4 Zcytor17 Based Proliferation in BAF3 Assay Using Alamar Blue

A. Construction of BaF3 Cells Expressing Zcytor17 Receptor, WSX-1Receptor and OSMR

BaF3 cells expressing the full-length zcytor17 receptor were constructedas per Example 2A above, using 30 μg of the zcytor17 expression vector,described in Example 3A. One exception is that in place of Geneticinselection, 2 μg/ml of Puromycin (ClonTech) was added to the transfectedcells in a T-162 flask to isolate the puromycin-resistant pool. The BaF3cells expressing the zcytor17 receptor mRNA were designated asBaF3/zcytor17. To obtain clones, Baf3/zcytor17 cells were counted in ahemocytometer and plated at 1 cell/well, 0.5 cell/well, 0.1 cell/well,and 0.01 cell/well in 96-well dishes. Fifteen clones were scaled up toT75 flasks, and five clones were assayed for zcytor17 expression. TotalRNA was isolated from cell pellets using a S.N.A.P.™ total RNA IsolationKit (InVitrogen). First-strand cDNA was synthesized using the proSTAR™First Strand RT-PCR kit, and then PCR with zcytor17 specific primersZC29,180 (SEQ ID NO:22) and ZC29,122 (SEQ ID NO:23) was performed toscreen the clones for expression of zcytor17. One clone,BaF3/zcytor17#15 was chosen to expand and transfect with the WSX-1expression vector.

BaF3 cells expressing zcytor17 and full-length WSX-1 were constructed asper Example 2A above, using 30 μg of the WSX-1 expression vectorWSX-1/pZp7Z (Example 3B) to electroporate the BaF3/zcytor17#15 cells.One exception is that in place of Geneticin selection, 200 μg/ml Zeocin(InVitrogen) was added to the transfected cells in a T-162 flask toisolate the zeocin-resistant pool. The BaF3 cells expressing zcytor17and WSX-1 were designated BaF3/zcytor17/hWSX-1. To obtain clones, poolsof Baf3/zcytor17/hWSX-1 cells were plated at limiting dilution in96-well plates. The resulting clones were expanded and total RNA wasisolated using a S.N.A.P.™ total RNA Isolation Kit (InVitrogen).First-strand cDNA was synthesized using the proSTAR™ First Strand RT-PCRkit, and then PCR with WSX-1 specific primers ZC9791 (SEQ ID NO:24) andZC9793 (SEQ ID NO:25) was used to screen the clones for expression ofWSX-1. One clone, BaF3/zcytor17/hWSX-1#5 was chosen to expand furtherand transfect with the OSMRbeta expression vector.

BaF3 cells expressing zcytor17, WSX-1 and full-length OSMRbeta wereconstructed as per Example 2A above, using 30 μg of the OSMRbetaexpression vector OSMR/pZp7NX described in Example 29 to electroporatethe BaF3/zcytor17/hWSX-1#5 cells. The BaF3 cells expressing zcytor17,WSX-1, and OSMRbeta mRNA were designated BaF3/zcytor17/WSX-1/OSMR. Toobtain clones, pools of BaF3/zcytor17/WSX-1/OSMRbeta cells were platedat limiting dilution in 96-well plates. Individual clones were expandedand total RNA was isolated using a S.N.A.P.™ total RNA Isolation Kit(InVitrogen). First-strand cDNA was synthesized using the proSTAR™ FirstStrand RT-PCR kit, and then PCR with OSMRbeta specific primers ZC40109(SEQ ID NO:26) and ZC40112 (SEQ ID NO:27) was used to screen the clonesfor expression of zcytor17, WSX-1, and OSMR. One clone,BaF3/zcytor17/WSX-1/OSMR#5 was selected and these cells were used toscreen for zcytor17lig as described below in Examples 5 and 6.

B. Construction of BaF3 Cells Expressing Zcytor17 Receptor and OSMR

BaF3 cells expressing the full-length zcytor17 receptor were constructedas per Example 2A above, using 30 μg of the zcytor17 expression vector,described in Example 3A. One exception is that in place of Geneticinselection, 2 μg/ml of Puromycin (ClonTech) was added to the transfectedcells in a T-162 flask to isolate the puromycin-resistant pool. The BaF3cells expressing the zcytor17 receptor mRNA were designated asBaF3/zcytor17. To obtain clones, pools of Baf3/zcytor17 cells wereplated at limiting dilution in 96-well plates. These clones wereexpanded in culture and total RNA was isolated using a S.N.A.P.™ totalRNA Isolation Kit (InVitrogen). First-strand cDNA was synthesized usingthe proSTAR™ First Strand RT-PCR kit, and then PCR was used to screenthe clones for expression of zcytor17. One clone, BaF3/zcytor17 #15 waschosen to expand and transfect with the OSMRbeta expression vector.

BaF3 cells expressing zcytor17 and full-length OSMRbeta were constructedas per Example 2A above, using 30 μg of the OSMRbeta expression vectorOSMR/pZp7NX (example 29) to electroporate the BaF3/zcytor17#15 cells.The BaF3 cells expressing zcytor17 and OSMRbeta mRNA were designatedBaF3/zcytor17/OSMR. These cells were used to screen for zcytor17lig asdescribed below in Example 5.

Example 5 Screening for Zcytor17lig Using BaF3/Zcytor17/WSX-1/OSMRbetaCells Using an Alamar Blue Proliferation Assay

A. Activation of CCRF-CEM and CCRF-HSB2 Cells to Test for Presence ofZcytor17lig

CCRF-CEM and CCRF-HSB2 cells were obtained from ATCC and stimulated inculture to produce conditioned media to test for the presence ofzcytor17lig activity as described below. The suspension cells wereseeded at 2×10⁵ cells/ml or 5×10⁵ cells/ml in RPMI-1640 mediasupplemented with 10% FBS, 2 mM L-glutamine (GibcoBRL), 1× PSN(GibcoBRL), and activated with 10 ng/ml Phorbol-12-myristate-13-acetate(PMA) (Calbiochem, San Diego, Calif.) and 0.5 μg/ml Ionomycin™(Calbiochem) for 24 or 48 hrs. The supernatant from the stimulated cellswas used to assay proliferation of the BaF3/zcytor17/WSX-1/OSMRbetacells or BaF3/zcytor17/OSMRbeta cells as described below.

B. Screening for Zcytor17lig Using BaF3/Zcytor17/WSX-1/OSMRbeta Cells orBaF3/Zcytor17/OSMRbeta Cells Using an Alamar Blue Proliferation Assay

BaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta cells werespun down and washed in mIL-3 free media. The cells were spun and washed3 times to ensure the removal of the mIL-3. Cells were then counted in ahemacytometer. Cells were plated in a 96-well format at 5000 cells perwell in a volume of 100 μl per well using the mIL-3 free media.

Proliferation of the BaF3/zcytor17/WSX-1/OSMRbeta cells orBaF3/zcytor17/OSMRbeta cells was assessed using conditioned media fromactivated CCRFCEM and CCRF-HSB2 cells (see Example 5A). Conditionedmedia was diluted with mIL-3 free media to 50%, 25%, 12.5%, 6.25%,3.125%, 1.5%, 0.75%, and 0.375% concentrations. 100 μl of the dilutedconditioned media was added to the BaF3/zcytor17/WSX-1/OSMRbeta cells orBaF3/zcytor17/OSMRbeta cells. The total assay volume is 200 μl. Theassay plates were incubated at 37° C., 5% CO₂ for 3-5 days at which timeAlamar Blue (Accumed, Chicago, Ill.) was added at 20 μl/well. Plateswere again incubated at 37° C., 5% CO₂ for 24 hours. Plates were read onthe Fmax™ plate reader (Molecular devices) as described above (Example2).

Results confirmed the proliferative response of theBaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta cells to afactor present in the activated CCRF-CEM and CCRF-HSB2 conditionedmedia. The response, as measured, was approximately 10-fold overbackground at the 25% concentration. The untransfected BaF3 cells didnot proliferate in response to this factor, nor did BaF3 cellstransfected with zcytor17 and WSX-1 (BaF3/zcytor17/WXS-1 cells), showingthat this factor was specific for Zcytor17/OSMRbeta orzcytor17/OSMRbeta/WSX-1 receptors. Moreover soluble zcytor17 receptordiminished this proliferative activity of zcytor17lig in theBaF3/zcytor17/WSX-1/OSMRbeta cells (see, Example 11). Similar resultsare expected in BaF3/zcytor17/OSMRbeta cells.

C. Human Primary Source Used to Isolate Zcytor17lig

One hundred milliliters blood draws were taken from each of six donors.The blood was drawn using 10×10 ml vacutainer tubes containing heparin.Blood was pooled from six donors (600 ml), diluted 1:1 in PBS, andseparated using a Ficoll-Paque® PLUS (Pharmacia Biotech). The isolatedprimary human cell yield after separation on the ficoll gradient was1.2×10⁹ cells.

Cells were suspended in 9.6 ml MACS buffer (PBS, 0.5% EDTA, 2 mM EDTA).1.6 ml of cell suspension was removed and 0.4 ml CD3 microbeads(Miltenyi Biotec, Auburn, Calif.) added. The mixture was incubated for15 min. at 4° C. These cells labeled with CD3 beads were washed with 30ml MACS buffer, and then resuspended in 2 ml MACS buffer.

A VS+ column (Miltenyi) was prepared according to the manufacturer'sinstructions. The VS+ column was then placed in a VarioMACS™ magneticfield (Miltenyi). The column was equilibrated with 5 ml MACS buffer. Theisolated primary human cells were then applied to the column. The CD3negative cells were allowed to pass through. The column was rinsed with9 ml (3×3 ml) MACS buffer. The column was then removed from the magnetand placed over a 15 ml falcon tube. CD3+ cells were eluted by adding 5ml MACS buffer to the column and bound cells flushed out using theplunger provided by the manufacturer. The incubation of the cells withthe CD3 magnetic beads, washes, and VS+ column steps (incubation throughelution) above were repeated five more times. The resulting CD3+fractions from the six column separations were pooled. The yield of CD3+selected human cells were 3×10⁸ total cells.

A sample of the pooled CD3+ selected human cells was removed forstaining and sorting on a fluorescent antibody cell sorter (FACS) toassess their purity. The human CD3+ selected cells were 91% CD3+ cells.

The human CD3+ selected cells were activated by incubating in RPMI+5%FBS+PMA 10 ng/ml and Ionomycin 0.5 μg/ml (Calbiochem) for 13 hours 37°C. The supernatant from these activated CD3+ selected human cells wastested for zcytor17lig activity as described below. Moreover, theactivated CD3+ selected human cells were used to prepare a cDNA library,as described in Example 6, below.

D. Testing Supernatant from Activated CD3+ Selected Human Cells forZcytor17lig Using BaF3/Zcytor17/WSX-1/OSMRbeta Cells and an Alamar BlueProliferation Assay

BaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta cells werespun down and washed in mIL-3 free media. The cells were spun and washed3 times to ensure the removal of the mIL-3. Cells were then counted in ahemacytometer. Cells were plated in a 96-well format at 5000 cells perwell in a volume of 100 μl per well using the mIL-3 free media.

Proliferation of the BaF3/zcytor17/WSX-1/OSMRbeta cells orBaF3/zcytor17/OSMRbeta cells were assessed using conditioned media fromactivated CD3+ selected human cells (see Example 5C) diluted with mIL-3free media to 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75%, 0.375% and 0.187%concentrations. 100 μl of the diluted conditioned media was added to theBaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta cells. Thetotal assay volume was 200 μl. The assay plates were incubated andassayed as described in Example 5B.

Results confirmed the proliferative response of theBaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta cells to afactor present in the activated CD3+ selected human Cell conditionedmedia. The response, as measured, was approximately 15-fold overbackground at the 25% concentration. The untransfected BaF3 cells didnot proliferate in response to this factor, nor did BaF3 cellstransfected with zcytor17 and WSX-1 (BaF3/zcytor17/WXS-1 cells), showingthat this factor is specific for Zcytor17/OSMRbeta orzcytor17/OSMRbeta/WSX-1 receptors.

Example 6 Cloning of Human Zcytor17lig from a Human CD3+ Selected CellLibrary

Screening of a primary human activated CD3+ selected cell cDNA libraryrevealed an isolated cDNA that is a novel member of the four-helixbundle cytokine family. This cDNA encoded the zcytor17lig. The cDNA wasidentified by screening for activity of the zcytor17lig using thezcytor17/WSX-1/OSM receptors.

A. The Vector for CD3+ Selected Library Construction

The vector for CD3+ selected library construction was pZP7NX. The pZP7NXvector was constructed as follows: The coding region for the DHFRselective marker in vector pZP7 was removed by DNA digestion with NcoIand PstI restriction enzymes (Boehringer Mannheim). The digested DNA wasrun on 1% agarose gel, cut out and gel purified using the Qiagen GelExtraction Kit (Qiagen) as per manufacturer's instructions. A DNAfragment representing the coding region of Zeocin selective marker wasamplified by PCR method with primers ZC13,946 (SEQ ID NO:28) andZC13,945 (SEQ ID NO:29), and pZeoSV2(+) as a template. There areadditional PstI and BclI restriction sites in primer ZC13,946 (SEQ IDNO:28), and additional NcoI and SfuI sites in primer ZC13,945 (SEQ IDNO:29). The PCR fragment was cut with PstI and NcoI restriction enzymesand cloned into pZP7 vector prepared by cleaving with the same twoenzymes and subsequent gel purification. This vector was named pZP7Z.Then the Zeocin coding region was removed by DNA digestion of vectorpZP7Z with BclI and SfuI restriction enzymes. The digested DNA was runon 1% agarose gel, cut out and gel purified, and then ligated with a DNAfragment of Neomycin coding region cut from pZem228 vector (deposited atthe American Type Culture Collection (ATCC), Manassas, Va.; ATCC DepositNo. 69446) with the same restriction enzymes (BclI and SfuI).

This new vector was named pZP7N, in which the coding region for DHFRselective marker was replaced by the coding region for a Neomycinselective marker from vector pZem228. A stuffer fragment including anXho1 site was added to pZP7N to create a vector suitable for highefficiency directional cloning of cDNA; this new vector was calledpZP7NX. To prepare the vector for cDNA, 20 μg of pZP7NX was digestedwith 20 units of EcoR1 (Life Technologies Gaithersberg, Md.) and 20units of Xho1 (Boehringer Mannheim Indianapolis, Ind.) for 5 hours at37° C., then 68° C. for 15 minutes. The digest was then run on a 0.8%low melt agarose 1×TAE gel to separate the stuffer from the vector. Thevector band was excised and digested with “beta-Agarase” (New EnglandBiolabs, Beverly, Mass.) following the manufacturer's recommendations.After ethanol precipitation the digested vector was resuspended in waterto 45 ng/ml in preparation for ligation of CD3+ selected cDNA librarydescribed below.

B. Preparation of Primary Human Activated CD3+ Selected Cell cDNALibrary

Approximately 1.5×10⁸ primary human CD3+ selected cells stimulated inionomycin/PMA were isolated by centrifugation after culturing at 37° C.for 13 hours (Example 5C). Total RNA was isolated from the cell pelletusing the “RNeasy Midi” kit from Qiagen, Inc. (Valencia, Calif.). mRNAwas isolated from 225 micrograms of total RNA using the “MPG mRNApurification kit” from CPG Inc. (Lincoln Park, N.J.). 3.4 micrograms ofmRNA was isolated and converted to double stranded cDNA using thefollowing procedure.

First strand cDNA from stimulated human CD3+ selected cells wassynthesized as follows. Nine μl Oligo d(T)-selected poly(A) CD3+ RNA ata concentration of 0.34 μg/μl and 1.0 μl of 1 μg/μl first strand primerZC18,698 (SEQ ID NO:30) containing an XhoI restriction site were mixedand heated at 65° C. for 4 minutes and cooled by chilling on ice. Firststrand cDNA synthesis was initiated by the addition of 9 μl of firststrand buffer (5× SUPERSCRIPT® buffer; (Life Technologies), 4 μl of 100mM dithiothreitol and 2 μl of a deoxynucleotide triphosphate solutioncontaining 10 mM each of dATP, dGTP, dTTP and 5-methyl-dCTP (PharmaciaBiotech Inc.) to the RNA-primer mixture. The reaction mixture wasincubated at 45° C. for 4 minutes followed by the addition of 8 μl of200 U/μl SuperscriptII®, RNase H— reverse transcriptase (Lifetechnologies). The reaction was incubated at 45° C. for 45 minutesfollowed by an incubation ramp of 1° C. every 2 minutes to 50° C. wherethe reaction was held for 10 minutes. To denature any secondarystructure and allow for additional extension of the cDNA the reactionwas then heated to 70° C. for 2 minutes then dropped to 55° C. for 4minutes after which 2 μl of SuperscriptII® RT was added and incubated anadditional 15 minutes followed by a ramp up to 70° C. 1 minute/1° C.Unincorporated nucleotides were removed from the cDNA by twiceprecipitating in the presence of 2 μg of glycogen carrier, 2.0 Mammonium acetate and 2.5 volume ethanol, followed by a 100 μl wash with70% ethanol. The cDNA was resuspended in 98 μl water for use in secondstrand synthesis.

Second strand synthesis was performed on the first strand cDNA underconditions that promoted first strand priming of second strand synthesisresulting in DNA hairpin formation. The second strand reaction contained98 μl of the first strand cDNA, 30 μl of 5× polymerase 1 buffer (100 mMTris: HCl, pH 7.5, 500 mM KCl, 25 mM MgCl₂, 50 mM (H₄)₂SO₄), 2 μl of 100mM dithiothreitol, 6 μl of a solution containing 10 mM of eachdeoxynucleotide triphosphate, 5 μl of 5 mM b-NAD, 1 μl of 3 U/μl E. coliDNA ligase (New England Biolabs Inc.) and 4 μl of 10 U/μl E. coli DNApolymerase 1 (New England Biolabs Inc.). The reaction was assembled atroom temperature and was incubated at room temperature for 2 minutesfollowed by the addition of 4 μl of 3.8 U/μl RNase H (LifeTechnologies). The reaction was incubated at 15° C. for two hoursfollowed by a 15 minute incubation at room temperature. 10 μl of 1M TRISpH7.4 was added to the reaction and extracted twice withphenol/chloroform and once with chloroform, the organic phases were thenback extracted with 50 μl of TE (10 mM TRIS pH 7.4, 1 mM EDTA), pooledwith the other aqueous and ethanol precipitated in the presence of 0.3 Msodium acetate. The pellet was washed with 100 μl 70% ethanol air driedand resuspended in 40 μl water.

The single-stranded DNA of the hairpin structure was cleaved using mungbean nuclease. The reaction mixture contained 40 μl of second strandcDNA, 5 μl of 10× mung bean nuclease buffer (Life technologies), 5 μl ofmung bean nuclease (Pharmacia Biotech Corp.) diluted to 1 U/μl in 1×mung bean nuclease buffer. The reaction was incubated at 37° C. for 45minutes. The reaction was terminated by the addition of 10 μl of 1 MTris: HCl, pH 7.4 followed by sequential phenol/chloroform andchloroform extractions as described above. Following the extractions,the cDNA was ethanol precipitated in the presence of 0.3 M sodiumacetate. The pellet was washed with 100 μl 70% ethanol air dried andresuspended in 38 μl water.

The resuspended cDNA was blunt-ended with T4 DNA polymerase. The cDNA,which was resuspended in 38 μl of water, was mixed with 12 μl 5× T4 DNApolymerase buffer (250 mM Tris:HCl, pH 8.0, 250 mM KCl, 25 mM MgCl₂), 2μl 0.1 M dithiothreitol, 6 μl of a solution containing 10 mM of eachdeoxynucleotide triphosphate and 2 μl of 1 U/μl T4 DNA polymerase(Boehringer Mannheim Corp.). After an incubation of 45 minutes at 15°C., the reaction was terminated by the addition of 30 μl TE followed bysequential phenol/chloroform and chloroform extractions and backextracted with 20 μl TE as described above. The DNA was ethanolprecipitated in the presence of 2 μl Pellet Paint™ (Novagen) carrier and0.3 M sodium acetate and was resuspended 11 μl of water.

EcoRI adapters were ligated onto the 5′ ends of the cDNA described aboveto enable cloning into an expression vector. 11 μl of cDNA and 4 μl of65 pmole/μl of Eco RI hemiphophorylated adaptor (Pharmacia Biotech Corp)were mixed with 5 μl 5× ligase buffer (Life Technologies), 2 μl of 10 mMATP and 3 μl of 1 U/μl T4 DNA ligase (Life Technologies), 1 μl 10×ligation buffer (Promega Corp), 9 μl water. The extra dilution with 1×buffer was to prevent the pellet paint from precipitating. The reactionwas incubated 9 hours in a water bath temperature ramp from 10° C. to22° C. over 9 hours, followed by 45 minutes at 25° C. The reaction wasterminated by incubation at 68° C. for 15 minutes.

To facilitate the directional cloning of the cDNA into an expressionvector, the cDNA was digested with XhoI, resulting in a cDNA having a 5′Eco RI cohesive end and a 3′ XhoI cohesive end. The XhoI restrictionsite at the 3′ end of the cDNA had been previously introduced using theZC18698 (SEQ ID NO:31) primer. Restriction enzyme digestion was carriedout in a reaction mixture containing 35 μl of the ligation mix describedabove, 6 μl of 10× H buffer (Boehringer Mannheim Corp.), 1 μl of 2 mg/mlBSA (Biolabs Corp.), 17 μl water and 1.0 μl of 40 U/μl XhoI (BoehringerMannheim). Digestion was carried out at 37° C. for 1 hour. The reactionwas terminated by incubation at 68° C. for 15 minutes followed byethanol precipitation, washing drying as described above andresuspension in 30 μl water.

The resuspended cDNA was heated to 65° C. for 5 minutes and cooled onice, 4 μl of 5× gel loading dye (Research Genetics Corp.) was added, thecDNA was loaded onto a 0.8% low melt agarose 1×TAE gel (SEA PLAQUE GTG™low melt agarose; FMC Corp.) and electrophoresed. The contaminatingadapters and cDNA below 0.6 Kb in length were excised from the gel. Theelectrodes were reversed, molten agarose was added to fill in the wells,the buffer was changed and the cDNA was electrophoresed untilconcentrated near the lane origin. The area of the gel containing theconcentrated cDNA was excised and placed in a microfuge tube, and theagarose was melted by heating to 65° C. for 15 minutes. Followingequilibration of the sample to 45° C., 2 μl of 1 U/μl Beta-agarase I(Biolabs, Inc.) was added, and the mixture was incubated for 90 min. at45° C. to digest the agarose. After incubation, 1 tenth volume of 3 M Naacetate was added to the sample, and the mixture was incubated on icefor 15 minutes. The sample was centrifuged at 14,000×g for 15 minutes atroom temperature to remove undigested agarose, the cDNA was ethanolprecipitated, washed in 70% ethanol, air-dried and resuspended in 40 μlwater.

To determine the optimum ratio of cDNA to vector several ligations wereassembled and electroporated. Briefly, 2 μl of 5× T4 ligase buffer (LifeTechnologies), 1 μl of 10 mM ATP, 1 μl pZP7NX digested with EcoR1-Xho1,1 μl T4 DNA ligase diluted to 0.25 u/μl (Life Technologies) water to 10μl and 0.5, 1,2 or 3 μl of cDNA were mixed in 4 separate ligations,incubated at 22° C. for 4 hours, 68° C. for 20 minutes, sodiumacetate-ethanol precipitated, washed, dried and resuspended in 10 μl. Asingle microliter of each ligation was electroporated into 40 μl DH10bElectroMax™ electrocompetent bacteria (Life Technologies) using a 0.1 cmcuvette (Biorad) and a Genepulser, pulse controller™ (Biorad) set to 2.5KV, 251 F, 200 ohms. These cells were immediately resuspended in 1 mlSOC broth (Manniatis et al. supra.) followed by 500 μL of 50%glycerol-SOC as a preservative. These “glycerol stocks” were frozen inseveral aliquots at −70° C. An aliquot of each was thawed and platedserially on LB-agar plates supplemented with ampicillin at 100 μg/ml.Colony numbers indicated that the optimum ratio of CD3+ cDNA to pZP7NXvector was 1 μl to 45 ng; such a ligation yielded 4.5 million primaryclones.

For the purpose of screening the library using a BaF3-basedproliferation assay (Example 5) glycerol stocks from above were dilutedinto liquid cultures of 100 or 250 clones per pool in deep wellmicrotiter plates, grown 24 hours at 37° C. with shaking and plasmidisolated using a Qiagen kit following the manufacturer's instructions.Such DNA was subsequently transfected into BHK cells, media conditioned72 hours, harvested and stored at −80° C., and subsequently placed on 5KBaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta cells for72 hours after which proliferation was assessed using an “Alamar blue”fluorescence assay (Example 5B and Example 2B).

Example 7 Expression Cloning of Human Zcytor17lig

The glycerol stocks from the activated human CD3+ selected cell library(Example 6) were added to Super Broth II™ (Becton Dickinson,Cockeysville, Md.)+0.1 mg/ml ampicillin (amp) at a concentration of 250cells per 800 microliters. The E. coli were allowed to equilibrate for24 hours at room temperature. At the time of inoculation, 400microliters was plated on LB+amp plates to determine the actual titer ofthe inoculation. After 24 hours the plates were counted and then thefinal concentration of the SuperBrothII™+E. coli was adjusted so thatthe final concentration was 250 cells per 1.2 ml. Three times 2 literswere inoculated for a total of 6 liters. The media were then plated into96-well deep well blocks (Qiagen). Plating was done on the 8-channelQ-Fill2™ dispenser (Genetix, Christchurch, Dorset, UK). The E. coli weregrown overnight at 37° C. shaking at 250 rotations/min. on a NewBrunswick Scientific Innova 4900 multi-tier environment shaker. The E.coli were spun out of solution at 3000 rpm, using a Beckman GS-6KRcentrifuge. These E. coli pellets were frozen at −20° C. or used freshbefore miniprepping the plasmid DNA. Each pellet contains approximately250 cDNA clones from the human CD3+ selected cell library.

These pools of 250 cDNA clones were then mini-prepped using QIAprep™ 96Turbo Miniprep kit (Qiagen). Plasmid DNA was eluted using 125 μl of TE(10 mM Tris pH 8, 1 mM EDTA). This plasmid DNA was then used totransfect BHK cells.

BHK Transfection

BHK cells were plated in 96-well tissue culture plates at a density of12,000 cells per well in a volume of 100 μl. per well. Culture media wasDMEM (GibcoBRL), 5% heat-inactivated fetal bovine serum, 2 mML-glutamine (GibcoBRL), 1× PSN (GibcoBRL), 1 mM NaPyruvate (GibcoBRL).

The following day, BHK cells were washed once with 100 μl SFA. SFA isserum-free media which is DMEM/F12 or DMEM (Gibco/BRL), 2 mM GlutaMax™(Gibco/BRL), 1 mM NaPyruvate, 10 μg/ml transferrin, 5 μg/ml insulin, 10μg/ml fetuin, 2 μg/ml selenium, 25 mM HEPES (Gibco/BRL), 100 μMnon-essential amino acids (Gibco/BRL).

A DNA/Lipofectamine™ mix was made as follows: 2.2 μl Lipofectamine™reagent (Gibco/BRL) was combined with 102.8 μl SFA at room temperature;approximately 5 μl of the plasmid DNA (200 ng/μl) was then added to theLipofectamine™/SFA to form the DNA/Lipofectamine™ mixture, which wasincubated at room temperature for 30 minutes. The SFA was removed fromthe BHK cells and the cells were incubated with 50 μl of theDNA/lipofectamine™ mix for 5 hours at 37° C. with 5% CO₂. Fifty μl ofthe DNA/Lipofectamine™ mixture was added to each of two wells of the BHKcells, so that transfections were done in duplicate.

After BHK cells were incubated with DNA/Lipofectamine™ mix for 5 hours,the DNA/Lipofectamine™ mix was removed and 100 μl culture media wasadded. Cells were incubated overnight, the media was removed andreplaced with 100 μl. culture media. After culturing cells for 48-72hours, conditioned media was removed, frozen at −80° C. for a minimum of20 minutes, thawed, and then 50 μl was assayed in the Baf3 proliferationassay, described in Example 5, to identify pools of 250 clones withligand activity.

Twenty 96-well plates were screened in a single assay. This representedapproximately 250 cDNAs/well or 480,000 cDNAs total. Of these,conditioned media from approximately 60 wells (representing 250 cDNAsper well) tested positive in the proliferation assay. One of thesepositive pools was chosen to break down and isolate a single cDNA thatwould encode the zcytor17lig. This was pool 62A12.

For pool 62A12, 1 μl. of DNA was used to transform ElectroMax™ DH10Bcells (Gibco/BRL) by electroporation. The transformants were plated onLB+amp (100 μg/ml) plates to give single colonies. From theelectroporated pool, 672 individual colonies were selected by toothpickinto seven 96-well plates containing 1.2 ml of SuperBrothII™ per well.These plates were numbered #62.1 through #62.7. These were culturedovernight and the plasmid DNA miniprepped as above. For all sevenplates, plasmid DNA from the breakdown plates was transfected into BHKcells and assayed by proliferation as above, except that transfectionswere not done in duplicate.

Two positive clones 62.6C7 and 62.6E9 were identified by activity from atotal of 672 clones. Plasmid DNA miniprepped from clone 62.6E9 wassequenced and a tentative identification was obtained, but a mixedsequence was obtained from this positive clones. To further isolate thezcytor17lig cDNA to a single clone, 1 μl of DNA from pool 62.6E9 wasused to electroporate DH10B cells and the transformants plated on LB+amp(100 μg/ml) plates to give single colonies. Plasmid DNA miniprepped fromseveral colonies was sequenced to give the exact DNA sequence. Thepolynucleotide sequence of zcytor17lig was full-length (SEQ ID NO:1) andits corresponding amino acid sequence is shown (SEQ ID NO:2).

Example 8 Construction of Mammalian Expression Vectors that ExpressZcytor17 Soluble Receptors: Zcytor17CEE, Zcytor17CFLG, Zcytor17CHIS andZcytor17-Fc4

A. Construction of Zcytor17 Mammalian Expression Vector ContainingZcytor17CEE, Zcytor17CFLG and Zcytor17CHIS

An expression vector was prepared for the expression of the soluble,extracellular domain of the zcytor17 polypeptide, pZp9zcytor17CEE, wherethe construct is designed to express a zcytor17 polypeptide comprised ofthe predicted initiating methionine and truncated adjacent to thepredicted transmembrane domain, and with a C-terminal Glu-Glu tag (SEQID NO:32).

An approximately 1500 bp PCR product was generated using ZC29,451 (SEQID NO:33) and ZC29,124 (SEQ ID NO:34) as PCR primers to add EcoRI andBamHI restriction sites. A human HPVS in-house cDNA library was used asa template and PCR amplification was performed as follows: 30 cycles at94° C. for 1 minute, 65° C. for 1 minute, 72° C. for 1.5 minutes, then72° C. for 7 minutes; 10° C. soak. The PCR reaction was ethanolprecipitated and digested with EcoRI and BamHI restriction enzymes. Thedigested PCR product was gel purified on a 1.0% agarose gel and theapproximately 1500 bp band excised. This band was then re-amplifiedusing identical primers with the following cycling: 30 cycles at 94° C.for 1 minute, 65° C. for 1 minute, 72° C. for 3 minutes, then 72° C. for7 minutes; 10° C. soak. The PCR reaction was ethanol precipitated anddigested with EcoRI and BamHI restriction enzymes. The digested PCRproduct was gel purified on a 1.0% agarose gel and the approximately1500 bp band excised. The excised DNA was subcloned into plasmid CEEpZp9that had been cut with EcoRI and BamHI, to generate plasmid with aGLU-GLU C-terminally tagged soluble receptor for zcytor17,zcytor17CEEpZp9. The extracellular domain in the zcytor17CEE cDNA inzcytor17CEEpZp9 has a silent mutation that changes the T to C atposition 1705 of SEQ ID NO:4 (encoding a Pro residue at residue 403 ofSEQ ID NO:5). As this mutation is silent, the zcytor17 cDNA inzcytor17CEEpZp9 encodes the polypeptide as shown in SEQ ID NO:5.Moreover, because of the construct used, a Gly-Ser residue pair isinserted C-terminal to the end of the soluble, extracellular domain ofzcytor17 and prior to the C-terminal Glu-Glu Tag (SEQ ID NO:32). Assuch, the tag at the C-terminus of the zcytor17 extracellular domain,was a Glu-Glu tag as shown in (SEQ ID NO:17). Plasmid CEEpZp9 is amammalian expression vector containing an expression cassette having themouse metallothionein-1 promoter, multiple restriction sites forinsertion of coding sequences, and a human growth hormone terminator.The plasmid also has an E. coli origin of replication, a mammalianselectable marker expression unit having an SV40 promoter, enhancer andorigin of replication, a DHFR gene and the SV40 terminator. Usingstandard molecular biological techniques zcytor17CEEpZp9 waselectroporated into DH10B competent cells (GIBCO BRL, Gaithersburg, Md.)according to manufacturer's direction and plated onto LB platescontaining 100 μg/ml ampicillin, and incubated overnight. Colonies werescreened by restriction analysis, or PCR from DNA prepared fromindividual colonies. The insert sequence of positive clones was verifiedby sequence analysis. A large scale plasmid preparation was done using aQIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.

The same process is used to prepare the zcytor17 soluble receptors witha C-terminal his tag, composed of 6 His residues in a row; and aC-terminal FLAG® tag (SEQ ID NO:36), zcytor17CFLAG. To construct theseconstructs, the aforementioned vector has either the HIS or the FLAG®tag in place of the glu-glu tag (e.g., SEQ ID NO:17; SEQ ID NO:32 or SEQID NO:35).

B. Mammalian Expression Construction of Soluble Human Zcytor17 Receptor:Zcytor17-Fc4

An expression vector, pEZE-2 hzcytor17/Fc4, was prepared to express aC-terminally Fc4 tagged soluble version of hzcytor17 (humanzcytor17-Fc4) in PF CHO cells. PF CHO cells are an in house CHO cellline adapted for growth in protein-free medium (ExCell 325 PF medium;JRH Biosciences). The in house CHO cell line was originally derived fromCHO DG44 cells (G. Urlaub, J. Mitchell, E. Kas, L. A. Chasin, V. L.Funanage, T. T. Myoda and J. L. Hamlin, “The Effect Of Gamma Rays at theDihydrofolate Reductase Locus: Deletions and Inversions,” Somatic Celland Molec. Genet., 12: 555-566 (1986). A fragment of zcytor17 cDNA thatincludes the polynucleotide sequence from extracellular domain of thezcytor17 receptor was fused in frame to the Fc4 polynucleotide sequence(SEQ ID NO:37) to generate a zcytor17-Fc4 fusion (SEQ ID NO:38 and SEQID NO:39). The pEZE-2 vector is a mammalian expression vector thatcontains the Fc4 polynucleotide sequence and a cloning site that allowsrapid construction of C-terminal Fc4 fusions using standard molecularbiology techniques.

A 1566 base pair fragment was generated by PCR, containing theextracellular domain of human zcytor17 and the first two amino acids ofFc4 (Glu and Pro) with FseI and BglII sites coded on the 5′ and 3′ ends,respectively. This PCR fragment was generated using primers ZC29,157(SEQ ID NO:40) and ZC29,150 (SEQ ID NO:41) by amplification from aplasmid containing the extracellular domain of human zcytor17(pZp9zcytor17CEE) (Example 8). The PCR reaction conditions were asfollows: 25 cycles of 94° C. for 1 minute, 60° C. for 1 minute, and 72°C. for 2 minutes; 1 cycle at 72° C. for 10 minutes; followed by a 4° C.soak. The fragment was digested with FseI and BglII restrictionendonucleases and subsequently purified by 1% gel electrophoresis andband purification using QiaQuick gel extraction kit (Qiagen). Theresulting purified DNA was ligated for 5 hours at room temperature intoa pEZE-2 vector previously digested with FseI and BglII containing Fc43′ of the FseI and BglII sites.

Two μl of the ligation mix was electroporated in 37 μl DH10Belectrocompetent E. coli (Gibco) according to the manufacturer'sdirections. The transformed cells were diluted in 400 μl of LB media andplated onto LB plates containing 100 μg/ml ampicillin. Clones wereanalyzed by restriction digests and positive clones were sent for DNAsequencing to confirm the sequence of the fusion construct. 1 μl of apositive clone was transformed into 37 μl of DH10B electrocompetent E.coli and streaked on a LB/amp plate. A single colony was picked fromthis streaked plate to start a 250 ml LB/amp culture that was then grownovernight at 37° C. with shaking at 250 rpm. This culture was used togenerate 750 μg of purified DNA using a Qiagen plasmid Maxi kit(Qiagen).

Example 9 Transfection and Expression of Zcytor17 Soluble ReceptorPolypeptides

BHK 570 cells (ATCC No. CRL-10314), DG-44 CHO, or other mammalian cellsare plated at about 1.2×10⁶ cells/well (6-well plate) in 800 μl ofappropriate serum free (SF) media (e.g., DMEM, Gibco/BRL High Glucose)(Gibco BRL, Gaithersburg, Md.). The cells are transfected withexpression plasmids containing zcytor17CEE, zcytor17CFLG, zcytor17CHISor zcytor17-Fc4 (Example 8), using Lipofectin™ (Gibco BRL), in serumfree (SF) media according to manufacturer's instruction. Single clonesexpressing the soluble receptors are isolated, screened and grown up incell culture media, and purified using standard techniques.

A. Mammalian Expression of Soluble Human Zcytor17CEE Receptor

BHK 570 cells (ATCC NO: CRL-10314) were plated in T-75 tissue cultureflasks and allowed to grow to approximately 50 to 70% confluence at 37°C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL High Glucose, (Gibco BRL,Gaithersburg, Md.), 5% fetal bovine serum, 1 mM L-glutamine (JRHBiosciences, Lenea, Kans.), 1 mM sodium pyruvate (Gibco BRL)). The cellswere then transfected with the plasmid containing zcytor17CEE (Example8) using Lipofectamine™ (Gibco BRL), in serum free (SF) mediaformulation (DMEM, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/mlfetuin, 1% L-glutamine and 1% sodium pyruvate). Ten μg of the plasmidDNA pZp9zcytor17CEE (Example 8) was diluted into a 15 ml tube to a totalfinal volume of 500 μl with SF media. 50 μl of Lipofectamine was mixedwith 450 μl of SF medium. The Lipofectamine mix was added to the DNA mixand allowed to incubate approximately 30 minutes at room temperature.Four ml of SF media was added to the DNA:Lipofectamine mixture. Thecells were rinsed once with 5 ml of SF media, aspirated, and theDNA:Lipofectamine mixture was added. The cells were incubated at 37° C.for five hours, and then 5 ml of DMEM/10% FBS media was added. The flaskwas incubated at 37° C. overnight after which time the cells were splitinto the selection media (DMEM/FBS media from above with the addition of1 μM methotrexate or 10 μM Methotrexate (Sigma Chemical Co., St. Louis,Mo.) in 150 mm plates at 1:2, 1:10, and 1:50. Approximately 10 dayspost-transfection, one 150 mm plate of 1 μM methotrexate resistantcolonies was trypsinized, the cells were pooled, and one-half of thecells were replated in 10 μM methotrexate; to further amplify expressionof the zcytor17CEE protein. A conditioned-media sample from this pool ofamplified cells was tested for expression levels using SDS-PAGE andWestern analysis.

B. Mammalian Expression of Soluble Human Zcytor17-Fc4 Receptor

Five replicates of 200 μg of pEZE-2hzcytor17Fc4 plasmid DNA (Example 8)were linearized by restriction digestion with FspI, a restriction enzymethat cuts once within the vector and does not disturb genes necessaryfor expression. 200 μg of CHO cell genomic DNA was added to eachreplicate as carrier DNA and then the DNA was precipitated by additionof 0.1 volumes of 3M Sodium Acetate pH 5.2 and 2.2 volumes ethanolfollowed by a 15 minute ice incubation and microcentrifugation at 4° C.The resulting DNA pellets were washed in 70% ethanol and air driedbefore being resuspended in 100 μl protein free (PF) CHO non-selectiongrowth media (21 g/L PF CHO Ex Cell 325/200 mM L-glutamine (Gibco)/100mM sodium pyruvate (Gibco)/1× HT Supplement (Gibco). Ten million PF CHOpassage 61 cells were added to the DNA in 600 μl of PF CHO non-selectiongrowth media and then electroporated in a Gene Pulser II Electroporationsystem (BioRad) using 950 μF capacitance and 300 Kv using a 0.4 cm gapGene Pulser (BioRad) electroporation cuveffe. All 5 replicates of theelectroporated cells were pooled and directly selected in —HT media (21g/L PF CHO Ex Cell 325/200 mM L-glutamine (Gibco)/100 mM sodium pyruvate(Gibco). Cells were selected for 15 days in —HT media before beingpassaged at 4×10⁵ ml into 50 nm MTX selection. Eight days later cellswere seeded at 3.5×10⁵ cells/ml into 200 mM MTX selection. After oneweek, cells were seeded at 4×10⁵ cells/ml into 1 μM MTX selection. Aftertwo weeks at 1 μM MTX, cells were seeded at 1×10⁶ cells/ml into 50 ml togenerate conditioned medium. The resulting 72 hour conditioned media wasanalyzed by probing western blots with an antibody generated againsthuman Ig. The cells produced hzcytor17/Fc4 protein at approximately 1mg/L.

C. Larger-Scale Mammalian Expression of Soluble Human Zcytor17-Fc4Receptor

Two hundred micrograms of pEZE-2hzcytor17Fc4 plasmid DNA (Example 8) waslinearized by restriction digestion with FspI, a restriction enzyme thatcuts once within the pEZE-2 vector and does not disturb genes necessaryfor expression. 200 μg of CHO genomic DNA (prepared in-house) was addedas carrier DNA and then the DNA was precipitated by addition of 0.1volumes of 3M Sodium Acetate pH 5.2 and 2.5 volumes ethanol followed bymicrocentrifugation at Room temperature. Five replicate DNA pellets weremade and transformed. The resulting DNA pellet was washed in 70% ethanoland air dried before being resuspended in 100 μl PF CHO non-selectiongrowth media (21 g/L PF CHO Ex Cell 325/200 mM L-glutamine (Gibco)/100mM sodium pyruvate (Gibco)/1× HT Supplement (Gibco). Ten million PF CHOcells were added to the DNA in 600 μl of PF CHO non-selection growthmedia and then electroporated in a Gene Pulser II Electroporation system(BioRad) using 950 μF capacitance and 300 volts using a 0.4 cm gap GenePulser (BioRad) electroporation cuvette. The electroporated cells werepooled and put directly into selection in —HT media (21 g/L PF CHO ExCell 325/200 mM L-glutamine (Gibco)/100 mM sodium pyruvate (Gibco).Cells were selected for 14 days in —HT media before being passaged at4×10⁵/ml into 50 nm MTX selection. Cells were amplified to 200 nM MTXand then to 1 uM MTX. The —HT, 50 nM, and 1 uM pools were seeded at1×10⁶ c/ml for 48 hours, and the resulting conditioned media wasanalyzed by probing western blots with an antibody generated againsthuman Ig.

Example 10 Purification of Zcytor17 Soluble Receptors from BHK 570 andCHO Cells

A. Transient Mammalian Expression and Purification of Soluble HumanZcytor17-Fc4 Receptor

pEZE-2hzcytor17Fc4 plasmid DNA (Example 11B) was introduced into 40 maxiplates of BHK cells using Lipofectamine (Gibco BRL) as described hereinand in manufacturer's instructions. Cells were allowed to recoverovernight, then were rinsed and refed with serum-free medium (SL7V4,made in-house). After 72 hours, the media was collected and filtered,and cells were refed with serum-free medium. After 72 hours, the mediawas again collected and filtered.

The serum-free conditioned media (2×1.5 L batches) from transientlytransfected BHK cells was pumped over a 1.5 ml Protein A-agarose columnin 20 mM Tris, pH 7.5, 0.5 M NaCl. The column was washed extensivelywith this buffer and then the bound protein was eluted with 1 ml of 0.2M glycine, pH 2.5, 0.5 M NaCl. The eluted protein was collected into 0.1ml of 2 M Tris, pH 8.5.

Aliquots were collected for SDS-polyacrylamide gel electrophoresis andthe bulk zcytor17-Fc was dialyzed overnight against PBS. The solublereceptor was sterile filtered and placed in aliquots at −80° C.

B. Purification of Zcytor17-Fc4

Recombinant carboxyl terminal Fc4 tagged zcytor17 (Example 8 and Example9) was produced from transfected CHO cells. The CHO transfection wasperformed using methods known in the art. Approximately five-liters ofconditioned media were harvested and sterile filtered using Nalgene 0.2μm filters.

Protein was purified from the filtered media by a combination of Poros50 protein A affinity chromatography (PerSeptive Biosystems, 1-5559-01,Framingham, Mass.) and Superdex 200 gel exclusion chromatography column(Amersham Pharmacia Biotech, Piscataway, N.J.). Culture medium wasdirectly loaded onto a 10×70 mm (5.5-ml bed volume) protein A affinitycolumn at a flow of about 3-10 ml minute. Following column washing forten column volumes of PBS, bound protein was eluted by five columnvolumes of 0.1 M glycine, pH 3.0 at 10 ml/minute). Fractions of 2 mleach were collected into tubes containing 100 μl of 2.0 M Tris, pH 8.0,in order to neutralize the eluted proteins. Samples from the affinitycolumn were analyzed by SDS-PAGE with coomassie staining and Westernblotting for the presence of zcytor17-Fc4 using human Ig-HRP.Zcytor17-Fc4-containing fractions were pooled and concentrated to 1-2 mlusing Biomax-30 concentrator (Millipore), and loaded onto a 20×580 mmSuperdex 200 gel filtration column. The fractions containing purifiedzcytor17-Fc4 were pooled, filtered through 0.2 μm filter, aliquoted into100 μl each, and frozen at −80° C. The concentration of the finalpurified protein was determined by BCA assay (Pierce, Rockford, Ill.).

C. SDS-PAGE and Western Blotting Analysis of Zcytor17/Fc4

Recombinant zcytor17-Fc4 was analyzed by SDS-PAGE (Nupage 4-12%,Invitrogen, Carlsbad, Calif.) with coomassie staining method and Westernblotting using human Ig-HRP. Either the conditioned media or purifiedprotein was electrophoresed using an Invitrogen Novex's Xcell IImini-cell, and transferred to nitrocellulose (0.2 mm; Invitrogen,Carlsbad, Calif.) at room temperature using Novex's Xcell II blot modulewith stirring according to directions provided in the instrument manual.The transfer was run at 500 mA for one hour in a buffer containing 25 mMTris base, 200 mM glycine, and 20% methanol. The filters were thenblocked with 10% non-fat dry milk in PBS for 10 minutes at roomtemperature. The nitrocellulose was quickly rinsed, then the humanIg-HRP antibody (1:2000) was added in PBS containing 2.5% non-fat drymilk. The blots were incubated for two hours at room temperature, orovernight at 4° C., with gentle shaking. Following the incubation, theblots were washed three times for 10 minutes each in PBS, then quicklyrinsed in H₂O. The blots were developed using commercially availablechemiluminescent substrate reagents (SuperSignal® ULTRA reagents 1 and 2mixed 1:1; reagents obtained from Pierce, Rockford, Ill.), and thesignal was captured using Lumi-Imager's Lumi Analyst 3.0 software(Boehringer Mannheim GmbH, Germany) for exposure times ranging from 10second to 5 minutes or as necessary.

The purified zcytor17-Fc4 appeared as a single band with either thecoomassie or silver staining at about 220 kDa under non-reducingconditions, and at about 120 kDa under reducing conditions, suggestingthe dimeric form of zcytor17-Fc4 under non-reducing conditions asexpected.

Example 11 Assay Using Zcytor17 Soluble Receptor Zcytor17-Fc4 SolubleReceptor in Competitive Inhibition Assay

BaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta cells werespun down and washed in mIL-3 free media. The cells were spun and washed3 times to ensure the removal of the mIL-3. Cells were then counted in ahemacytometer. Cells were plated in a 96-well format at 5000 cells perwell in a volume of 100 μl per well using the mIL-3 free media.

Both conditioned media from the CCRF-CEM and CCRF-HSB2 cell activationand the human CD3+ selected cells, described in Example 5, were added inseparate experiments at 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75%, 0.375%,and 0.187% concentrations, with or without zcytor17 soluble receptors(Zcytor17-Fc4; See, Example 9 and Example 10) at 1-10 μg/ml. The totalassay volume was 200 μl.

The assay plates were incubated at 37° C., 5% CO₂ for 3-5 days at whichtime Alamar Blue (Accumed) was added at 20 μl well. Plates were againincubated at 37° C., 5% CO₂ for 16-24 hours. Plates were read on theFmax™ plate reader (Molecular Devices) as described in Example 2.Results demonstrated partial inhibition of cell growth with zcytor17-Fc4soluble receptor at 10 μg/ml, confirming that the factor in each samplewas specific for the zcytor17 receptor.

Titration curves, diluting out the soluble receptor, or soluble receptorheterodimers and trimers comprising zcytor17 receptor (e.g.,zcytor17/OSMR, zcytor17/WSX-1, or zcytor17/OSMR/WSX-1, or other Class Icytokine receptor subunits) are also run using the above stated assay todetermine whether zcytor17 receptors are able to completely inhibitgrowth, for example, at low or physiologic concentrations.

Example 12 Secretion Trap Assay

A secretion trap assay is used to test the binding of the zcytor17lig toreceptors comprising zcytor17 receptor, such as the zcytor17 receptor orreceptor heterodimers and trimers comprising zcytor17 receptor (e.g.,zcytor17/OSMR, zcytor17/WSX-1, or zcytor17/OSMR/WSX-1, or other Class Icytokine receptor subunits). Zcytor17lig plasmid DNA is transfected intoCOS cells, and used to assess binding of the zcytor17lig to receptorscomprising zcytor17 receptor by secretion trap as described below.

A. COS Cell Transfections

The COS cell transfection is performed as follows: Mix about 800 ng ofzcytor17lig cDNA and 5 μl Lipofectamine™ in 92 μl serum free DMEM media(55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5 mginsulin, 1 g selenium and 5 mg fetuin in 500 ml DMEM), incubate at roomtemperature for 30 minutes and then add 400 μl serum free DMEM media.Add this 500 μL mixture onto 1.5×10⁵ COS cells/well plated on 12-welltissue culture plate and incubate for 5 hours at 37° C. Add 500 μL 20%FBS DMEM media (100 ml FBS, 55 mg sodium pyruvate and 146 mg L-glutaminein 500 ml DMEM) and incubate overnight.

B. Secretion Trap Assay

The secretion trap is performed as follows: Media is rinsed off cellswith PBS and then fixed for 15 minutes with 1.8% Formaldehyde in PBS.Cells are then washed with TNT (0.1M Tris-HCL, 0.15M NaCl, and 0.05%Tween-20 in H₂O), and permeated with 0.1% Triton-X in PBS for 15minutes, and again washed with TNT. Cells are blocked for 1 hour withTNB (0.1M Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (EN RenaissanceTSA-Direct Kit) in H₂O), and washed again with TNT. If using thebiotinylated receptor protein, the cells are blocked for 15 minuteincubations with Avidin and then Biotin (Vector Labs) washing in-betweenwith TNT. Depending on which soluble receptor is used, the cells areincubated for 1 hour in TNB with: (A) 1-3 μg/ml zcytor17 solublereceptor zcytor17-Fc4 fusion protein (Example 10); (B) 1-3 μg/mlzcytor17/OSMRbeta soluble receptor protein; (C) 1-3 μg/ml zcytor17/WSX-1soluble receptor protein; or (D) 1-3 μg/ml zcytor17/OSMR/WSX-1 solublereceptor protein. Cells are then washed with TNT. Depending on whichsoluble receptor is used (e.g., if labeled with an Fc4 tag (SEQ IDNO:37), C-terminal FLAG tag (SEQ ID NO:36), or CEE tag (SEQ ID NO:32;SEQ ID NO:35)), cells are incubated for another hour with: (A) 1:200diluted goat-anti-human Ig-HRP (Fc specific); (B) 1:1000 diluted M2-HRP;(C) 1:1000 diluted anti-GluGlu antibody-HRP; or (D) 1:300 dilutedstreptavidin-HRP (NEN kit) in TNB, for example. Again cells are washedwith TNT.

Positive binding is detected with fluorescein tyramide reagent diluted1:50 in dilution buffer (EN kit) and incubated for 4-6 minutes, andwashed with TNT. Cells are preserved with Veetashield Mounting Media(Vector Labs Burlingame, Calif.) diluted 1:5 in TNT. Cells arevisualized using a FITC filter on fluorescent microscope.

Example 13 Chromosomal Assignment and Placement of the Gene Sequence forthe Zcytor17lig

The zcytor17lig gene sequence was mapped to human chromosome 12 usingthe commercially available version of the “Stanford G3 Radiation HybridMapping Panel” (Research Genetics, Inc., Huntsville, Ala.). The“Stanford G3 RH Panel” contains DNA from each of 83 radiation hybridclones of the whole human genome, plus two control DNAs (the RM donorand the A3 recipient). A publicly available WWW server (e.g., StandfordUniversity) allows chromosomal localization of markers and genes.

For the mapping of the zcytor17lig gene sequence with the “Stanford G3RH Panel”, 20 μl reactions were set up in a 96-well microtiter platecompatible for PCR (Stratagene, La Jolla, Calif.) and used in a“RoboCycler Gradient 96” thermal cycler (Stratagene). Each of the 95 PCRreactions consisted of 2 μl 10× PCR reaction buffer (Qiagen, Inc.,Valencia, Calif.), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, FosterCity, Calif.), 1 μl sense primer, ZC41,458 (SEQ ID NO:42), 1 μlantisense primer, ZC41,457 (SEQ ID NO:43), 2 μl “RediLoad” (ResearchGenetics, Inc., Huntsville, Ala.), 0.1 μl Qiagen HotStarTaq DNAPolymerase (5 units/μl), 25 ng of DNA from an individual hybrid clone orcontrol and distilled water for a total volume of 20 μl. The reactionswere overlaid with an equal amount of mineral oil and sealed. The PCRcycler conditions were as follows: an initial 1 cycle 15 minutedenaturation at 95° C., 35 cycles of a 45 second denaturation at 95° C.,1 minute annealing at 53° C. and 1 minute and 15 seconds extension at72° C., followed by a final 1 cycle extension of 7 minutes at 72° C. Thereactions were separated by electrophoresis on a 2% agarose gel (EMScience, Gibbstown, N.J.) and visualized by staining with ethidiumbromide.

The results showed linkage of the zcytor17lig gene sequence to thechromosome 12 marker SHGC-83339 with a LOD score of >11 and at adistance of 17 cR_(—)10000 from the marker. This marker positionszcytor17lig gene in the 12q24.31 chromosomal region.

Example 14 Identification and Cloning of Murine Zcytor17lig

A. Identification of Full Length Murine Zcytor17lig

Using the human zcytor17lig peptide sequence (SEQ ID NO:2) to query anin house DNA database, a murine cDNA, Genbank Accession No. AK005939,was identified as a potential partial sequence for the murinezcytor17lig. The AK005939 cDNA sequence was used to query a databasecontaining murine genomic fragments. A genomic contig of the murinezcytor17lig was assembled (SEQ ID NO:76). Prediction of coding potentialon this genomic fragment with the program Genscan revealed a likely cDNAsequence, with the same gene structure as the human zcytor17lig. Amurine cDNA sequence is represented in SEQ ID NO:10, and correspondingpolypeptide sequence is shown in SEQ ID NO:11.

B. Cloning of Mouse Zcytor17lig from a Mouse Testis cDNA Library by PCR

Based on the genomic sequence (SEQ ID NO:76), two PCR primers weredesigned and used to identify a cDNA source of mouse zcytor17lig by PCR.These Primers ZC41498 (SEQ ID NO:86) and ZC41496 (SEQ ID NO:87) weredesigned to the putative 5′ and 3′ untranslated regions of the mousesequences (SEQ ID NO:76 and SEQ ID NO:10). Several cDNA sources werescreened by PCR, including Marathon-ready cDNAs (Clontech) and aliquotsof locally made cDNA libraries. Products were visualized on 1% agarosegels. Bands of the expected size were observed in reactions utilizing amouse testis cDNA library template. These PCR reactions weresuccessfully performed in approximately 50 μl volumes with or without10% DMSO, using pfu turbo polymerase (Stratagene) according to themanufacturer's recommendations; with an additional application of a waxhot-start employing hot start 50 s (Molecular Bioproducts, Inc. SanDiego, Calif.). PCR thermocycling was performed with a single cycle of94° C. for 4 min; followed by 40 cycles of 94° C.: 30 seconds, 48° C.:30 seconds, 72° C.: 50 seconds; with additional final 72° C. extensionfor 7 minutes. The two PCR reactions were pooled and purified using lowmelt agarose and Gelase agarose digesting enzyme (Epicenter, Inc.Madison, Wis.) according to the manufacturer's recommendations.

DNA sequence determination of these PCR products revealed a murinezcytor17 cDNA sequence (SEQ ID NO:90) which comprised an ORF identicalto SEQ ID NO:10, confirming that SEQ ID NO:10 encoded the mousezcytor17lig polypeptide. PCR primers, ZC41583 (SEQ ID NO:88) and ZC41584(SEQ ID NO:89), were then used to add FseI and AscI restriction sitesand a partial Kozak sequence to the mcytor17lig open reading frame andtermination codon (SEQ ID NO:92). A Robocycler 40 thermocycler(Stratagene) was used to run a temperature gradient of annealingtemperatures and cycling as follows. Pfu turbo polymerase (Stratagene)was applied as described above, but only in 10% DMSO. Cycling wasperformed with a single cycle of 94° C. for 4 min; followed by 20 cyclesof 94° C.: 30 seconds, 65° C. to 51° C. gradient: 30 seconds, 72° C.: 1minute; and a single 72° C. extension for 7 minutes. The template forthis second thermocycling reaction was 1 μl of the initial gel-purifiedmcytor17lig PCR product, above. Resulting PCR product from the threelowest temperature reactions were pooled and gel purified using theGelase (Epicenter) method described above. This purified mzcytor17ligwas digested with FseI and AscI and ligated into a pZP7X vector modifiedto have FseI and AscI sites in its cloning site. Plasmid pZP7X is amammalian expression vector containing an expression cassette having themouse metallothionein-1 (MT-1) promoter, multiple restriction sites forinsertion of coding sequences, and a human growth hormone terminator.The plasmid also has an E. coli origin of replication, a mammalianselective marker expression unit having an SV40 promoter, enhancer andorigin of replication, a DHFR gene, and the SV40 terminator. The clonedmurine cDNA sequence is represented in SEQ ID NO:90, and correspondingpolypeptide sequence is shown in SEQ ID NO:91 (which is identical to SEQID NO:11).

Example 15 Isolation of Mouse Zcytor17lig cDNA Clone from an ActivatedMouse Spleen Library

A. Murine Primary Source Used to Isolate Mouse Zcytor17lig

Mouse spleens from Balb/C mice, are collected and mashed betweenfrosted-end slides to create a cell suspension. The isolated primarymouse cell yield is expected to be about 6.4×10⁸ cells prior toselection described below.

The spleen cells are suspended in 9.6 ml MACS buffer (PBS, 0.5% EDTA, 2mM EDTA). 1.6 ml of cell suspension is removed and 0.4 ml CD90 (Thy1.2)microbeads (Miltenyi Biotec) added. The mixture is incubated for 15 min.at 4° C. These cells labeled with CD90 beads are washed with 30 ml MACSbuffer, and then resuspended in 2 ml MACS buffer.

A VS+ column (Miltenyi) is prepared according to the manufacturer'sinstructions. The VS+ column is then placed in a VarioMACS™ magneticfield (Miltenyi). The column is equilibrated with 5 ml MACS buffer. Theisolated primary mouse cells are then applied to the column. The CD90negative cells are allowed to pass through. The column is rinsed with 9ml (3×3 ml) MACS buffer. The column is then removed from the magnet andplaced over a 15 ml falcon tube. CD90+ cells are eluted by adding 5 mlMACS buffer to the column and bound cells flushed out using the plungerprovided by the manufacturer. The incubation of the cells with the CD90magnetic beads, washes, and VS+ column steps (incubation throughelution) above are repeated once more. The resulting CD90+ fractionsfrom the 2 column separations are pooled. The yield of CD90+ selectedmouse spleen cells are expected to be about 1×10⁸ total cells.

A sample of the pooled CD90+ selected mouse cells is removed forstaining and sorting on a fluorescent antibody cell sorter (FACS) toassess their purity. A PE-conjugated hamster anti-mouse CD3ε antibody(PharMingen) is used for staining and sorting the CD90+ selected cells.The mouse CD90+ selected cells should be about 93% CD3+ cells,suggesting the cells are 93% T-cells.

The murine CD90+ selected cells are activated by incubating 3×10⁶cells/ml in RPMI+5% FBS+PMA 10 ng/ml and Ionomycin 0.5 μg/ml(Calbiochem) for overnight at 37° C. The supernatant from theseactivated CD90+ selected mouse cells is tested for zcytor17lig activityas described below. Moreover, the activated CD90+ selected mouse cellsare used to prepare a cDNA library, as described in Example 16, below.

Example 16 Cloning of Mouse Zcytor17lig from a Mouse CD90+ Selected CellLibrary

Screening of a primary mouse activated CD90+ selected cell cDNA librarycan reveal isolated cDNA that is a novel member of the four-helix bundlecytokine family that would encode the mouse ortholog of the humanzcytor17lig. The cDNA is identified by hybridization screening.

A. The Vector for CD90+ Selected Library Construction

The vector, pZP7N is used for CD3+ selected library construction (SeeExample 6A)

B. Preparation of Primary Mouse Activated CD90+ Selected Cell cDNALibrary

Approximately 1.5×10⁸ primary mouse CD90+ selected cells stimulated inionomycin/PMA (Example 15) are isolated by centrifugation. Total RNA isisolated from the cell pellet, and converted to double stranded cDNA asdescribed in Example 6B. This DNA is subsequently transfected into BHKcells, as described in Example 6B, and proliferation is assessed usingan “Alamar blue” fluorescence assay (Example 2B).

For the purpose of screening the library by secretion trap cloning, acomplex, amplified form of the library is needed to transfect COS-7cells. 4.8 million clones are plated on 110 15 cm LB-agar platessupplemented with 100 μg/ml ampicillin, 10 μg/ml methicillin. Aftergrowing the plates overnight at 37° C. the bacteria are harvested byscraping and pelleted. Plasmid DNA is extracted from the pelletedbacteria using a Nucleobond-giga™ (Clonetech) following themanufacturer's instructions. This plasmid is then used to transfectCOS-7 cells on slides and screened using the secretion trap techniquedescribed below (Example 17).

C. Screening the Activated Mouse cDNA Library

Approximately 5×0⁵ clones are plated on 10 LB/Amp Maxi plates. Thecolonies are lifted, denatured, neutralized, and cross-linked using thestandard procedure (Sambrook, J. et al. supra.). Fifty nanograms of the300 bp 5′ RACE PCR fragment (Example 14) is labeled with ³²P usingPrime-Itr RmT random primer labeling kit (Stratagene). The 10 filtersare hybridized with this labeled probe at 65° C. overnight usingExpressHyb™ Hybridization Solution (Clontech). The filters are thenwashed sequentially at 60° C. for 1 hour three times with 0.2×SSC (30 mMNaCl, 3 mM sodium citrate, pH 7.0), 0.1% SDS; and then at 65° C. for 1hour. The filters are exposed at −80° C. overnight, and the X-ray filmare developed. Agar plugs containing the positive colonies are pulled,and the clones plated on 10-cm LB/Amp plates. The colonies are thenfilter-lifted and hybridized again following the same proceduredescribed above. Single DNA clones are isolated and sequenced usingstandard methods, to identify the mouse cDNA.

Example 17 Mouse Zcytor17lig does not Bind to Human Zcytor17 SolubleReceptor in Secretion Trap Assay

The DNA for mouse clone mzcytor17lig/pZP7 was transfected into COScells, and the binding of zcytor17 comprising soluble receptors (humanzcytor17 soluble receptor zcytor17-Fc4 (Example 10), or soluble receptorheterodimers (zcytor17/WSX-1 or BaF3/zcytor17/OSMRbeta), to thetransfected COS cells were tested by a secretion trap assay (Example12). The assay confirmed that the mouse zcytor17lig does not bind tohuman zcytor17 soluble receptor.

The COS cell transfection was performed as per Example 12 using about0.7 μg mouse zcytor17lig cDNA (Example 16) in 3 μl.

The secretion trap was performed as per example 12 using, for example, 1μg/ml zcytor17 soluble receptor Fc4 fusion protein (Example 10) (orzcytor17 comprising soluble receptor heterodimers as described herein)in TNB, and 1:200 diluted goat-anti-human Ig-HRP (Fc specific) in TNBfor the detectable antibody. Positive binding of the soluble humanzcytor17 receptor to the prepared fixed cells was not detected withfluorescein tyramide reagent as per Example 12. Cells were preserved andvisualized according to Example 12.

Results indicated that the mouse zcytor17lig does not bind to humanzcytor17 soluble receptor (or zcytor17 comprising soluble receptorheterodimers as described herein).

Example 18 Expression of Mouse Zcytor17lig in Mammalian Cells

Mammalian Expression of Mouse Zcytor17lig

BHK 570 cells (ATCC No: CRL-10314) were plated in 10 cm tissue culturedishes and allowed to grow to approximately 20% confluence overnight at37° C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL High Glucose media;Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum (Hyclone, Logan,Utah), 1 mM L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mM sodiumpyruvate (Gibco BRL). The cells were then transfected with the plasmidmzcytor17lig/pZP7X (Example 14) using a mammalian stable Lipofectamine(GibcoBRL) transfection kit according to the manufacturer'sinstructions.

One day after transfection, the cells were split 1:10 and 1:20 into theselection media (DMEM/FBS media with the addition of 1 μM methotrexate(Sigma Chemical Co., St. Louis, Mo.)) in 150 mm plates. The media on thecells was replaced with fresh selection media at day 5post-transfection. Approximately 10 days post-transfection, methotrexateresistant colonies were trypsinized and the cells pooled and plated intolarge-scale culture flasks. Once the cells were grown to approximately90% confluence, they were rinsed with PBS three times, and cultured withserum-free ESTEP2 media (DMEM (Gibco BRL), 0.11 g/l Na Pyruvate, 3.7 g/lNaHCO₃, 2.5 mg/l insulin, 5 mg/l transferrin, pH7.0) conditioned media.The conditioned media was collected three days later, and put into aBaF3 proliferation assay using Alamar Blue, described in Example 19below.

Example 19 Mouse Zcytor17lig does not Activate Human Zcytor17 Receptorin BaF3 Assay Using Alamar Blue

Proliferation of BaF3/zcytor17, BaF3/zcytor17/OSMRbeta andBaF3/zcytor17/WSX-1 cells (Example 4, and 5B) was assessed usingserum-free conditioned media from BHK cells expressing mouse zcytor17lig(Example 18). BaF3/Zcytor17, BaF3/zcytor17/OSMRbeta andBaF3/zcytor17/WSX-1 cells were spun down, washed and plated in mIL-3free media as described in Example 5B. Conditioned media from BHK cellsexpressing mouse zcytor17lig (Example 18) was diluted with mIL-3 freemedia to 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375%concentrations. The proliferation assay was performed as per Example 5B.The results of this assay were negative, indicating that mousezcytor17lig does not activate human zcytor17, zcytor17/OSMRbeta, orzcytor17/WSX-1 receptor complexes.

Example 20 Human Zcytor17lig Activates Human, Zcytor17/OSMRbetaReceptor, in Luciferase Assay

A. Construction of BaF3/KZ134/Zcytor17 Cell Line

The KZ134 plasmid was constructed with complementary oligonucleotidesZC12,749 (SEQ ID NO:44) and ZC12,748 (SEQ ID NO:45) that contain STATtranscription factor binding elements from 4 genes, which includes amodified c-fos Sis inducible element (m67SIE, or hSIE) (Sadowski, H. etal., Science 261:1739-1744, 1993), the p21 SIE1 from the p21 WAF1 gene(Chin, Y. et al., Science 272:719-722, 1996), the mammary gland responseelement of the β-casein gene (Schmitt-Ney, M. et al., Mol. Cell. Biol.11:3745-3755, 1991), and a STAT inducible element of the Fcg RI gene,(Seidel, H. et al., Proc. Natl. Acad. Sci. 92:3041-3045, 1995). Theseoligonucleotides contain Asp718-XhoI compatible ends and were ligated,using standard methods, into a recipient firefly luciferase reportervector with a c-fos promoter (Poulsen, L. K. et al., J. Biol. Chem.273:6229-6232, 1998) digested with the same enzymes and containing aneomycin selectable marker. The KZ134 plasmid was used to stablytransfect BaF3 cells, using standard transfection and selection methods,to make the BaF3/KZ134 cell line.

A stable BaF3/KZ134 indicator cell line, expressing the full-lengthzcytor17 receptor or zcytor17/OSMRbeta receptor was constructed as perExample 4. Clones were diluted, plated and selected using standardtechniques. Clones were screened by luciferase assay (see Example 20B,below) using the human zcytor17lig conditioned media or purifiedzcytor17lig protein (see Example 35, below) as an inducer. Clones withthe highest luciferase response (via STAT luciferase) and the lowestbackground were selected. Stable transfectant cell lines were selected.The cell lines were called BaF3/KZ134/zcytor17 orBaF3/KZ134/zcytor17/OSMRbeta depending on the receptors transfected intothe cell line.

Similarly, BHK cell lines were also constructed using the methoddescribed herein, and were used in luciferase assays described herein.The cell lines were called BHK/KZ134/zcytor17 orBHK/KZ134/zcytor17/OSMRbeta depending on the receptors transfected intothe cell line.

B. Human Zcytor17lig Activates Human Zcytor17 Receptor inBaF3/KZ134/Zcytor17/OSMRbeta or BHK/KZ134/Zcytor17/OSMRbeta LuciferaseAssay

BaF3/KZ134/zcytor17 and BaF3/KZ134/zcytor17/OSMRbeta cells were spundown and washed in mIL-3 free media. The cells were spun and washed 3times to ensure removal of mIL-3. Cells were then counted in ahemacytometer. Cells were plated in a 96-well format at about 30,000cells per well in a volume of 100 μl per well using the mIL-3 freemedia. The same procedure was used for untransfected BaF3/KZ134 cellsfor use as a control in the subsequent assay. BHK/KZ134/zcytor17 orBHK/KZ134/zcytor17/OSMRbeta cells were plated in a 96-well format at15,000 cells per well in 100 μl media. Parental BHK/KZ134 cells wereused as a control.

STAT activation of the BaF3/KZ134/Zcytor17,BaF3/KZ134/zcytor17/OSMRbeta, BHK/KZ134/zcytor17, orBHK/KZ134/zcytor17/OSMRbeta cells was assessed using (1) conditionedmedia from BHK570 cells transfected with the human zcytor17lig (Example7), (2) conditioned media from BHK570 cells transfected with the mousezcytor17lig (Example 18), (3) purified human zcytor17lig (Example 35),or (4) mIL-3 free media to measure media-only control response.Conditioned media was diluted with RPMI mIL-3 free media to 50%, 25%,12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375% concentrations. Purifiedhuman zcytor17lig was diluted to a concentration of 1200, 600, 300, 150,75, 37.5, 18.75, or 9.4 pM. 100 μl of the diluted conditioned media orprotein was added to the BaF3/KZ134/Zcytor17,BaF3/KZ134/zcytor17/OSMRbeta, BHK/KZ134/zcytor17, orBHK/KZ134/zcytor17/OSMRbeta cells. The assay using the conditioned mediawas done in parallel on untransfected BaF3/KZ134 or BHK/KZ134 cells as acontrol. The total assay volume was 200 μl. The assay plates wereincubated at 37° C., 5% CO₂ for 24 hours at which time the BaF3 cellswere pelleted by centrifugation at 2000 rpm for 10 min., and the mediawas aspirated and 25 μl of lysis buffer (Promega) was added. For the BHKcell lines, the centrifugation step was not necessary as the cells areadherant. After 10 minutes at room temperature, the plates were measuredfor activation of the STAT reporter construct by reading them on aluminometer (Labsystems Luminoskan, model RS) which added 40 μl ofluciferase assay substrate (Promega) at a five second integration.

The results of this assay confirmed that the STAT reporter response ofthe BaF3/KZ134/zcytor17/OSMRbeta and BHK/KZ134/zcytor17/OSMRbeta cellsto the human zcytor17lig when compared to either the BaF3/KZ134/zcytor17cells, the BHK/KZ134/zcytor17 cells or the untransfected BaF3/KZ134 orBHK/KZ134 control cells, showed that the response was mediated throughthe zcytor17/OSMRbeta receptors. The results also showed that the mousezcytor17lig does not activate the STAT reporter assay through the humanreceptor complex.

Example 21 Mouse Zcytor17lig is Active in Mouse Bone Marrow Assay

A. Isolation of Non-Adherent Low Density Marrow Cells:

Fresh mouse femur aspirate (marrow) is obtained from 6-10 week old maleBalb/C or C57BL/6 mice. The marrow is then washed with RPMI+10% FBS(JRH, Lenexa Kans.; Hyclone, Logan Utah) and suspended in RPMI+10% FBSas a whole marrow cell suspension. The whole marrow cell suspension isthen subjected to a density gradient (Nycoprep, 1.077, Animal; GibcoBRL) to enrich for low density, mostly mononuclear, cells as follows:The whole marrow cell suspension (About 8 ml) is carefully pipeted ontop of about 5 ml Nycoprep gradient solution in a 15 ml conical tube,and then centrifuged at 600×g for 20 minutes. The interface layer,containing the low density mononuclear cells, is then removed, washedwith excess RPMI+10% FBS, and pelleted by centrifugation at 400×g for5-10 minutes. This pellet is resuspended in RPMI+10% FBS and plated in aT-75 flask at approximately 10⁶ cells/ml, and incubated at 37° C. 5% CO₂for approximately 2 hours. The resulting cells in suspension areNon-Adherent Low Density (NA LD) Marrow Cells.

B. 96-Well Assay

NA LD Mouse Marrow Cells are plated at 25,000 to 45,000 cells/well in 96well tissue culture plates in RPMI+10% FBS+1 ng/mL mouse Stem CellFactor (mSCF) (R&D Systems, Minneapolis, Minn.), plus 5% conditionedmedium from one of the following: (1) BHK 570 cells expressing mousezcytor17lig (Example 18), (2) BHK 570 cells expressing human zcytor17lig(Example 7), or (3) control BHK 570 cells containing vector and notexpressing either Ligand. These cells are then subjected to a variety ofcytokine treatments to test for expansion or differentiation ofhematopoietic cells from the marrow. For testing, the plated NA LD mousemarrow cells are subjected to human Interleukin-15 (hIL-15) (R&DSystems), or one of a panel of other cytokines (R&D Systems). Serialdilution of hIl-15, or the other cytokines, are tested, with 2-foldserial dilution from about 50 ng/ml down to about 0.5 ng/mlconcentration. After 8 to 12 days the 96-well assays are scored for cellproliferation by Alamar blue assay as described in Example 5B.

C. Results from the 96-Well NA LD Mouse Marrow Assay

Conditioned media from the BHK cells expressing both mouse and humanzcytor17lig can promote the expansion of a population of hematopoieticcells either alone or in synergy with other cytokines in the NA LD mousemarrow in comparison to control BHK conditioned medium. The populationhematopoietic cells expanded by the mouse zcytor17lig with or withoutother cytokines, and those hematopoietic cells expanded by the humanzcytor17lig with or without other cytokines, are further propagated incell culture. These hematopoietic cells are stained with a Phycoerythrinlabeled anti-Pan NK cell antibody (PharMingen) and subjected to flowcytometry analysis, which demonstrated that the expanded cells stainedpositively for this natural killer (NK) cell marker. Similarly, otherspecific hematopoietic cell markers can be used to determine expansionof, for example, CD4+ or CD8+ T-cells, other T-cell populations,B-cells, and other immune cell markers.

The same 96-well assay is run, using fresh human marrow cells boughtfrom Poietic Technologies, Gaithersburg, Md. Again, a positive resultshows that zcytor17lig alone or in synergy with other cytokines, themouse and human zcytor17lig can expand a hematopoietic cell populationthat is stained positively for specific cell markers as disclosed above.

Example 22 Constructs for Generating Zcytor17lig Transgenic Mice

Construct for expressing human zcytor17lig from the MT-1 promoterOligonucleotides are designed to generate a PCR fragment containing aconsensus Kozak sequence and the human zcytor17lig coding region. Theseoligonucleotides are designed with an FseI site at the 5′ end and anAscI site at the 3′ end to facilitate cloning into (a) pMT12-8, ourstandard transgenic vector, or (b) pKFO51, a lymphoid-specifictransgenic vector (Example 22B).

PCR reactions are carried out with about 200 ng human zcytor17ligtemplate (SEQ ID NO:1) and oligonucleotides designed to amplify thefull-length or active portion of the zcytor17lig. PCR reactionconditions are determined using methods known in the art. PCR productsare separated by agarose gel electrophoresis and purified using aQiaQuick™ (Qiagen) gel extraction kit. The isolated, correct sized DNAfragment is digested with FseI and AscI (Boerhinger-Mannheim), ethanolprecipitated and ligated into pMT12-8 previously digested with FseI andAscI. The pMT12-8 plasmid, designed for expressing a gene of interest inliver and other tissues in transgenic mice, contains an expressioncassette flanked by 10 kb of MT-15′ DNA and 7 kb of MT-1 3′ DNA. Theexpression cassette comprises the MT-1 promoter, the rat insulin IIintron, a polylinker for the insertion of the desired clone, and thehuman growth hormone (hGH) poly A sequence.

About one microliter of each ligation reaction is electroporated intoDH10B ElectroMax™ competent cells (GIBCO BRL, Gaithersburg, Md.)according to manufacturer's direction and plated onto LB platescontaining 100 μg/ml ampicillin, and incubated overnight. Colonies arepicked and grown in LB media containing 100 μg/ml ampicillin. MiniprepDNA is prepared from the picked clones and screened for the humanzcytor17lig insert by restriction digestion with EcoRI alone, or FseIand AscI combined, and subsequent agarose gel electrophoresis. Maxiprepsof the correct pMT-human zcytor17lig are performed. A SalI fragmentcontaining with 5′ and 3′ flanking sequences, the MT-1 promoter, the ratinsulin II intron, human zcytor17lig cDNA and the hGH poly A sequence isprepared to be used for microinjection into fertilized murine oocytes.Microinjection and production of transgenic mice are produced asdescribed in Hogan, B. et al. Manipulating the Mouse Embryo, 2^(nd) ed.,Cold Spring Harbor Laboratory Press, NY, 1994.

B. Construct for Expressing Human Zcytor17lig from the Lymphoid-SpecificEμLCK Promoter

Oligonucleotides are designed to generate a PCR fragment containing aconsensus Kozak sequence and the human zcytor17lig coding region. Theseoligonucleotides are designed with an FseI site at the 5′ end and anAscI site at the 3′ end to facilitate cloning into pKFO51, alymphoid-specific transgenic vector.

PCR reactions are carried out with about 200 ng human zcytor17ligtemplate (SEQ ID NO:1) and oligonucleotides designed to amplify thefull-length or active portion of the zcytor17lig. A PCR reaction isperformed using methods known in the art. The isolated, correct sizedDNA fragment is digested with FseI and AscI (Boerhinger-Mannheim),ethanol precipitated and ligated into pKFO51 previously digested withFseI and AscI. The pKFO51 transgenic vector is derived from p1026X(Iritani, B. M., et al., EMBO J. 16:7019-31, 1997) and contains the Tcell-specific lck proximal promoter, the B/T cell-specificimmunoglobulin μ heavy chain enhancer, a polylinker for the insertion ofthe desired clone, and a mutated hGH gene that encodes an inactivegrowth hormone protein (providing 3′ introns and a polyadenylationsignal).

About one microliter of each ligation reaction is electroporated,plated, clones picked and screened for the human zcytor17lig insert byrestriction digestion as described above. A correct clone ofpKFO51-zcytor17lig is verified by sequencing, and a maxiprep of thisclone is performed. A NotI fragment, containing the lck proximalpromoter and immunoglobulin μ enhancer (EμLCK), zcytor17lig cDNA, andthe mutated hGH gene is prepared to be used for microinjection intofertilized murine oocytes.

C. Construct for Expressing Mouse Zcytor17lig from the EF1alpha Promoter

Primers ZC41,498 (SEQ ID NO:86) and ZC41,496 (SEQ ID NO:87) were used toPCR a mouse testis cDNA library template. These PCR reactions weresuccessfully performed in approximately 50 μl volumes with or without10% DMSO, using pfu turbo polymerase (Stratagene) according to themanufacturer's recommendations; with an additional application of a waxhot-start employing hot start 50 s (Molecular Bioproducts, Inc. SanDiego, Calif.). PCR thermocycling was performed with a single cycle of94° C. for 4 min; followed by 40 cycles of 94° C.: 30 seconds, 48° C.:30 seconds, 72° C.: 50 seconds; with additional final 72° C. extensionfor 7 minutes. The two PCR reactions were pooled and purified using lowmelt agarose and Gelase agarose digesting enzyme (Epicenter, Inc.Madison, Wis.) according to the manufacturer's recommendations.

DNA sequenced PCR products revealed a murine zcytor17 cDNA sequence (SEQID NO:90) which comprised an ORF identical to SEQ ID NO:10, confirmingthat SEQ ID NO:10 encoded the mouse zcytor17lig polypeptide. PCRprimers, ZC41583 (SEQ ID NO:88) and ZC41584 (SEQ ID NO:89), were thenused to add FseI and AscI restriction sites and a partial Kozak sequenceto the mcytor17lig open reading frame and termination codon (SEQ IDNO:92). A Robocycler 40 thermocycler (Stratagene) was used to run atemperature gradient of annealing temperatures and cycling as follows.Pfu turbo polymerase (Stratagene) was applied as described above, butonly in 10% DMSO. Cycling was performed with a single cycle of 94° C.for 4 min; followed by 20 cycles of 94° C.: 30 seconds, 65° C. to 51° C.gradient: 30 seconds, 72° C.: 1 minute; and a single 72° C. extensionfor 7 minutes. The template for this second thermocycling reaction was 1μl of the initial gel-purified mcytor17lig PCR product, above. ResultingPCR product from the three lowest temperature reactions were pooled andgel purified using the Gelase (Epicenter) method described above. Thispurified fragment was then digested with FseI and AscI and ligated intoa pZP7X vector modified to have FseI and AscI sites in its cloning site.This was sent to sequencing to confirm the correct sequence. The clonedmurine cDNA sequence is represented in SEQ ID NO:90, and correspondingpolypeptide sequence is shown in SEQ ID NO:91 (which is identical to SEQID NO:11).

The isolated, correct sized DNA fragment digested with FseI and AscI

(Boerhinger-Mannheim) was subcloned into a plasmid containing EF1alphapromoter previously digested with FseI and AscI. Maxipreps of thecorrect EF1alpha mouse zcytor17lig were performed. The expressioncassette contains the EF1alpha promoter (with a deleted FseI site), theEF1alpha intron, SUR IRES like site to facilitate expression, apolylinker flanked with rat insulin II sites on the 5′end which addsFseI PmeI AscI sites for insertion of the desired clone, and the humangrowth hormone (hGH) poly A sequence. A 7.5 kb NotI fragment containingthe EF1alpha promoter expression cassette and mouse zcytor17lig wasprepared to be used for microinjection into fertilized murine oocytes.The EF1alpha plsdmid was obtained from Louis-Marie of the Laboratoire deDifferenciation Cellulaire, as described in Taboit-Dameron et al., 1999,Transgenic Research 8:223-235.

D. Construct for Expressing Mouse Zcytor17lig from the Lymphoid-SpecificEμLCK Promoter

Oligonucleotides were designed to generate a PCR fragment containing aconsensus Kozak sequence and the mouse zcytor17lig coding region. Theseoligonucleotides were designed with an FseI site at the 5′ end and anAscI site at the 3′ end to facilitate cloning into pKFO51 (see Example22B, above).

The isolated, correct sized zcytor17lig DNA fragment used in EF1alphaconstructs, digested with FseI and AscI (Boerhinger-Mannheim), wassubcloned into a plasmid containing pKFO51, a lymphoid-specifictransgenic vector. The pKFO51 transgenic vector is derived from p1026X(Iritani, B. M., et al., EMBO J. 16:7019-31, 1997) and contains the Tcell-specific lck proximal promoter, the B/T cell-specificimmunoglobulin μ heavy chain enhancer, a polylinker for the insertion ofthe desired clone, and a mutated hGH gene that encodes an inactivegrowth hormone protein (providing 3′ introns and a polyadenylationsignal). A 6.5 kb NotI fragment, containing the lck proximal promoterand immunoglobulin μ enhancer (EμLCK), mouse zcytor17lig cDNA, and themutated hGH gene was prepared to be used for microinjection intofertilized murine oocytes (Example 41).

Example 23 Construction of Mammalian Expression Vectors that ExpressZcytor17lig-CEE

A. Construction of ZCytor17Lig-CEE/pZMP21

An expression plasmid containing all or part of a polynucleotideencoding human zCytor17lig was constructed via homologous recombination.The plasmid was called zCytor17Lig-CEE/pZMP21.

The construction of zCytor17Lig-CEE/pZMP21 was accomplished bygenerating a zCytor17Lig-CEE fragment (SEQ ID NO:95) using PCRamplification. The DNA template used for the production of thezCytor17Lig-CEE fragment was zCytor17Lig/pZP7nx. The primers used forthe production of the zCytor17Lig-CEE fragment were: (1) ZC41607 (SEQ IDNO:97) (sense sequence), which includes from the 5′ to the 3′ end: 28 bpof the vector flanking sequence (5′ of the insert) and 21 bpcorresponding to the 5′ sequence of zCytor17Lig; and (2) ZC41605 (SEQ IDNO:98) (antisense sequence), which includes from the 5′ to the 3′ end:37 bp of the vector flanking sequence (3′ of the insert), 3 bp of thestop codon, 21 bp encoding a C-terminal EE tag, and 21 bp correspondingto the 3′ end of zCytor17Lig sequence. The fragment resulting from theabove PCR amplification is a copy of the template zCytor17Lig with theaddition of a C-terminal EE tag, yielding a final productzCytor17Lig-CEE.

PCR reactions were run as follows: To a 100 μl final volume was added:10 μl of 10× Taq Polymerase Reaction Buffer with 15 mM MgCl (Gibco), 1μl of Taq DNA Polymerase (5 units/μl, Gibco), 3 μl of 10 mM dNTPs, 78 μldH2O, 3 μl of a 20 pmol/μl stock of primer ZC41607 (SEQ ID NO:97) 3 μlof a 20 pmol/μl stock of primer ZC41605 (SEQ ID NO:98), and 2 μl of a0.13 μg/μl stock of zCytor17lig template DNA. A volume equal to 50 μl ofmineral oil was added to the mixture. The reaction was heated to 94° C.for 5 minutes, followed by 35 cycles at 94° C. for 1 minute; 55° C. for2 minutes; 72° C. for 3 minutes; followed by a 10 minute extension at72° C. and held at 4° C. until the reaction was collected.

The plasmid pZMP21 was restriction digested with BglII enzyme, cleanedwith a QiaQuick PCR Purification Kit (Qiagen) using a microcentrifugeprotocol, and used for recombination with the PCR fragment. PlasmidpZMP21 was constructed from pZMP20 which was constructed from pZP9(deposited at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, and is designated No. 98668) withthe yeast genetic elements taken from pRS316 (deposited at the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, and designated No. 77145), an IRES element from poliovirus,and the extracellular domain of CD8, truncated at the carboxyl terminalend of the transmembrane domain. PZMP21 is a mammalian expression vectorcontaining an expression cassette having the MPSV promoter,immunoglobulin signal peptide intron, multiple restriction sites forinsertion of coding sequences, a stop codon and a human growth hormoneterminator. The plasmid also has an E. coli origin of replication, amammalian selectable marker expression unit having an SV40 promoter,enhancer and origin of replication, a DHFR gene, the SV40 terminator, aswell as the URA3 and CEN-ARS sequences required for selection andreplication in S. cerevisiae.

Fifty microliters of competent yeast cells (S. cerevisiae) wereindependently combined with 100 ng of cut plasmid, 5 μl of previouslydescribed PCR mixture, and transferred to a 0.2 cm electroporationcuvette. The yeast/DNA mixture was electropulsed at 0.75 kV (5 kV/cm), ∞ohms, 25 μF. Each cuvette had 600 μl of 1.2 M sorbitol added, and theyeast was plated in one 100 μl aliquot and one 300 μl aliquot onto twoURA-D plates and incubated at 30° C. After about 72 hours, the Ura+yeast transformants from a single plate were resuspended in 1 ml H₂O andspun briefly to pellet the yeast cells. The cell pellet was resuspendedin 500 μl of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mMTris, pH 8.0, 1 mM EDTA). The 500 μl of the lysis mixture was added toan Eppendorf tube containing 300 μl acid washed 600 μm glass beads and300 μl phenol-chloroform, vortexed for 1 minute intervals two or threetimes, followed by a 5 minute spin in a Eppendorf centrifuge at maximumspeed. Three hundred microliters of the aqueous phase was transferred toa fresh tube, and the DNA precipitated with 600 μl 100% ethanol (EtOH),followed by centrifugation for 10 minutes at 4° C. The DNA pellet wasthen washed with 500 μl 70% EtOH, followed by centrifugation for 1minute at 4° C. The DNA pellet was resuspended in 30 μl H₂O.

Transformation of electrocompetent E. coli cells (MC1061) was done with5 μl of the yeast DNA prep and 50 μl of MC1061 cells. The cells wereelectropulsed at 2.0 kV, 25 μF and 400 ohms(Ω). Followingelectroporation, 600 μl SOC (2% Bacto' Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO₄, 20 mM glucose) was added. The electroporated E. coli cells wereplated in a 200 μl and a 50 μl aliquot on two LB AMP plates (LB broth(Lennox), 1.8% Bacto Agar (Difco), 100 mg/L Ampicillin). The plates wereincubated upside down for about 24 hours at 37° C. ThreeAmpicillin-resistant colonies were selected at random and submitted forsequence analysis of the insert. Large scale plasmid DNA was isolatedfrom a sequence-confirmed clone using the Qiagen Maxi kit (Qiagen)according to manufacturer's instructions.

B. Mammalian Expression of Human Zcytor17lig

Full-length zCytor17Lig protein was produced in BHK cells transfectedwith zcytor17Lig-CEE/pZMP21 (Example 23A). BHK 570 cells (ATCCCRL-10314) were plated in T75 tissue culture flasks and allowed to growto approximately 50 to 70% confluence at 37° C., 5% CO₂, in growth media(SL7V4, 5% FBS, 1% pen/strep). The cells were then transfected withzCytor17Lig-CEE/pZMP21 by liposome-mediated transfection (usingLipofectamine™; Life Technologies), in serum free (SF) media (SL7V4).The plasmid (16 μg) was diluted into 1.5 ml tubes to a total finalvolume of 640 μl with SF media. Thirty-five microliters of the lipidmixture was mixed with 605 μl of SF medium, and the resulting mixturewas allowed to incubate approximately 15 minutes at room temperature.Five milliliters of SF media was then added to the DNA:lipid mixture.The cells were rinsed once with 10 ml of PBS, the PBS was decanted, andthe DNA:lipid mixture was added. The cells are incubated at 37° C. forfive hours, then 15 ml of media (SL7V4, 5% FBS, 1% pen/strep) was addedto each plate. The plates were incubated at 37° C. overnight, and theDNA:lipid media mixture was replaced with selection media (SL7V4, 5%FBS, 1% pen/strep, 1 μM methotrexate) the next day. Approximately 10days post-transfection, methotrexate-resistant colonies from the T75transfection flask were trypsinized, and the cells were pooled andplated into a T-162 flask and transferred to large-scale culture.

Example 24 Expression of Zcytor17 Soluble Receptor in E. coli

A. Construction of Expression Vector pCMH01 that ExpressesHuzcytor17/MBP-6H Fusion Polypeptide

An expression plasmid containing a polynucleotide encoding a zcytor17soluble receptor fused C-terminally to maltose binding protein (MBP) wasconstructed via homologous recombination. The fusion polypeptidecontains an N-terminal approximately 388 amino acid MBP portion fused toany of the zcytor17 soluble receptors described herein. A fragment ofzcytor17 cDNA (SEQ ID NO:4) was isolated using PCR as described herein.Two primers were used in the production of the zcytor17 fragment in astandard PCR reaction: (1) one containing about 40 bp of the vectorflanking sequence and about 25 bp corresponding to the amino terminus ofthe zcytor17, and (2) another containing about 40 bp of the 3′ endcorresponding to the flanking vector sequence and about 25 bpcorresponding to the carboxyl terminus of the zcytor17. Two μl of the100 μl PCR reaction was run on a 1.0% agarose gel with 1×TBE buffer foranalysis, and the expected approximately fragment was seen. Theremaining PCR reaction was combined with the second PCR tube andprecipitated with 400 μl of absolute ethanol. The precipitated DNA usedfor recombining into the Sma1 cut recipient vector pTAP170 to producethe construct encoding the MBP-zcytor17 fusion, as described below.

Plasmid pTAP170 was derived from the plasmids pRS316 and pMAL-c2. Theplasmid pRS316 is a Saccharomyces cerevisiae shuttle vector (Hieter P.and Sikorski, R., Genetics 122:19-27, 1989). pMAL-C2 (NEB) is an E. coliexpression plasmid. It carries the tac promoter driving MalE (geneencoding MBP) followed by a His tag, a thrombin cleavage site, a cloningsite, and the rrnB terminator. The vector pTAP170 was constructed usingyeast homologous recombination. 100 ng of EcoR1 cut pMAL-c2 wasrecombined with 1 μg Pvu1 cut pRS316, 1 μg linker, and 1 g Sca1/EcoR1cut pRS316. The linker consisted of oligos zc19,372 (SEQ ID NO:172) (100pmole): zc19,351 (SEQ ID NO:173) (1 pmole): zc19,352 (SEQ ID NO:174) (1pmole), and zc19,371 (SEQ ID NO:175) (100 pmole) combined in a PCRreaction. Conditions were as follows: 10 cycles of 94° C. for 30seconds, 50° C. for 30 seconds, and 72° C. for 30 seconds; followed by4° C. soak. PCR products were concentrated via 100% ethanolprecipitation.

One hundred microliters of competent yeast cells (S. cerevisiae) werecombined with 10 μl of a mixture containing approximately 1 μg of thehuman zcytor17 insert, and 100 ng of SmaI digested pTAP170 vector, andtransferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixturewas electropulsed at 0.75 kV (5 kV/cm), infinite ohms, 25 μF. To eachcuvette was added 600 μl of 1.2 M sorbitol. The yeast was then plated intwo 300 μl aliquots onto two-URA D plates and incubated at 30° C.

After about 48 hours, the Ura+yeast transformants from a single platewere picked, DNA was isolated, and transformed into electrocompetent E.coli cells (e.g., MC1061, Casadaban et. al. J. Mol. Biol. 138, 179-207),and plated on MM/CA+KAN 25 μg/L plates (Pryor and Leiting, ProteinExpression and Purification 10:309-319, 1997) using standard procedures.Cells were grown in MM/CA with 25 μg/ml Kanomyacin for two hours,shaking, at 37° C. One ml of the culture was induced with 1 mM IPTG. Twoto four hours later the 250 μl of each culture was mixed with 250 μlacid washed glass beads and 250 μl Thorner buffer with 5% OME and dye(8M urea, 100 mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Sampleswere vortexed for one minute and heated to 65° C. for 10 minutes. 20 μlwere loaded per lane on a 4%-12% PAGE gel (NOVEX). Gels were run in1×MES buffer. The positive clones were designated pCMH01 and subjectedto sequence analysis.

One microliter of sequencing DNA was used to transform strain BL21. Thecells were electropulsed at 2.0 kV, 25 μF and 400 ohms. Followingelectroporation, 0.6 ml MM/CA with 25 μg/L Kanomyacin. Cells were grownin MM/CA and induced with ITPG as described above. The positive cloneswere used to grow up for protein purification of the huzcytor17/MBP-6Hfusion protein using standard techniques.

B. Purification of Huzcytor17/MBP-6H Soluble Receptor from E. coliFermentation

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for the purification of recombinanthuzcytor17/MBP-6H soluble receptor polypeptide. E. coli cells containingthe pCMH01 construct and expressing huzcytor17/MBP-6H soluble receptorpolypeptide were constructed using standard molecular biology methodsand cultured in SuperBroth II (12 g/L Casien, 24 g/L Yeast Extract, 11.4g/L di-potassium phosphate, 1.7 g/L Mono-potassium phosphate; BectonDickenson, Cockeysville, Md.). The resulting cells were harvested andfrozen in 0.5% glycerol. Twenty grams of the frozen cells were used forprotein purification.

Thawed cells were resuspended in 500 mL Amylose Equilibration buffer (20mM Tris, 100 mM NaCl, pH 8.0). A French Press cell breaking system(Constant Systems Ltd., Warwick, UK) with a temperature setting of −7°C. to −10° C. and 30K PSI was used to lyse the cells. The resuspendedcells were checked for breakage by A₆₀₀ readings before and aftercycling through the French Press. The lysed cell suspension was pelletedat 10,000 G for 30 minutes. Supernatant was harvested from the debrispellet for protein purification.

Twenty-five milliliters of Amylose resin (New England Biolabs, Beverly,Mass.) was poured into a Bio-Rad, 2.5 cm D×10 cm H glass column. Thecolumn was packed and equilibrated by gravity with 10 column volumes(CVs) of Amylose Equilibration buffer. The harvested cell supernatantwas batch loaded to the Amylose resin, overnight with rocking. Theloaded resin was returned to the glass column, washed with 10 CVsAmylose Equilibration buffer, and eluted by gravity with −2 CVs AmyloseElution buffer (Amylose Equilibration buffer, 10 mM Maltose, FlukaBiochemical, Switzerland). Ten 5 ml fractions were collected over theelution profile and assayed for absorbance at 280 and 320 nM. TheAmylose resin was regenerated with 1 CV of distilled H₂O, 5 CVs of 0.1%(w/v) SDS (Sigma), 5 CVs of distilled H₂O, 5 CVs of AmyloseEquilibration buffer, and finally 1 CV of Amylose Storage buffer(Amylose Equilibration buffer, 0.02% (w/v) Sodium Azide). Theregenerated resin was stored at 4° C.

Elution profile fractions of interest were pooled and dialyzed in a 10Kdialysis chamber (Slide-A-Lyzer, Pierce Immunochemical) against 4changes of 4 L PBS pH 7.4 (Sigma) over an 8 hour time period. Followingdialysis, the material harvested represented the purifiedhuzcytor17/MBP-6H polypeptide. The purified huzcytor17/MBP-6Hpolypeptide was filter sterilized and analyzed via SDS-PAGE Coomassiestaining for an appropriate molecular weight product. The concentrationof the huzcytor17/MBP-6H polypeptide was determined by BCA analysis tobe 0.76 mg/ml.

Purified huzcytor17/MBP-6H polypeptide was appropriately formulated forthe immunizaton of rabbits and sent to R & R Research and Development(Stanwood, Wash.) for polyclonal antibody production (Example 25,below).

Example 25 Human Zcytor17 Receptor Polyclonal Antibody

A. Preparation and Purification

Polyclonal antibodies were prepared by immunizing 2 female New Zealandwhite rabbits with the purified recombinant protein huzcytor17/MBP-6H(Example 24). The rabbits were each given an initial intraperitoneal(IP) injection of 200 μg of purified protein in Complete Freund'sAdjuvant followed by booster IP injections of 100 μg protein inIncomplete Freund's Adjuvant every three weeks. Seven to ten days afterthe administration of the second booster injection (3 total injections),the animals were bled and the serum was collected. The animals were thenboosted and bled every three weeks.

The huzcytor17/MBP-6H specific rabbit serum was pre-adsorbed of anti-MBPantibodies using a CNBr-SEPHAROSE 4B protein column (Pharmacia LKB) thatwas prepared using 10 mg of non-specific purified recombinant MBP-fusionprotein per gram of CNBr-SEPHAROSE. The huzcytor17/MBP-6H-specificpolyclonal antibodies were affinity purified from the pre-adsorbedrabbit serum using a CNBr-SEPHAROSE 4B protein column (Pharmacia LKB)that was prepared using 10 mg of the specific antigen purifiedrecombinant protein huzcytor17/MBP-6H. Following purification, thepolyclonal antibodies were dialyzed with 4 changes of 20 times theantibody volume of PBS over a time period of at least 8 hours. Humanzcytor17-specific antibodies were characterized by ELISA using 500 ng/mlof the purified recombinant protein huzcytor17/MBP-6H as antibodytarget. The lower limit of detection (LLD) of the rabbitanti-huzcytor17/MBP-6H affinity purified antibody is 500 μg/ml on itsspecific purified recombinant antigen huzcytor17/MBP-6H.

B. SDS-PAGE and Western Blotting Analysis of Rabbit Anti-Human ZcytoR17MBP-6H Antibody

Rabbit anti-human ZcytoR17 MBP-6H antibody was tested by SDS-PAGE(NuPage 4-12%, Invitrogen, Carlsbad, Calif.) with coomassie stainingmethod and Western blotting using goat anti-rabbit IgG-HRP. Eitherpurified protein (200-25 ng) or conditioned media containing zcytor17was electrophoresed using an Invitrogen Novex's Xcell II mini-cell, andtransferred to nitrocellulose (0.2 mm; Invitrogen, Carlsbad, Calif.) atroom temperature using Novex's Xcell blot module with stirring accordingto directions provided in the instrument manual. The transfer was run at300 mA for one hour in a buffer containing 25 mM Tris base, 200 mMglycine, and 20% methanol. The filter was then blocked with Western Abuffer (in house, 50 mM Tris, pH 7.4, 5 mM EDTA, pH 8.0, 0.05% IgepalCA-630, 150 mM NaCl, and 0.25% gelatin) overnight with gentle rocking at4° C. The nitrocellulose was quickly rinsed, then the rabbit anti-humanzcytoR17 MBP-6H (1:1000) was added in Western A buffer. The blot wasincubated for 1.5 hours at room temperature with gentle rocking. Theblot was rinsed 3 times for 5 minutes each in Western A, then goatanti-rabbit IgG HRP antibody (1:1000) was added in Western A buffer. Theblot was incubated for 1.25 hours at room temperature with gentlerocking. The blot was rinsed 3 times for 5 minutes each in Western A,then quickly rinsed in H₂O. The blot was developed using commerciallyavailable chemiluminescent substrate reagents (ECLWestern blottingdetection reagents 1 and 2 mixed 1:1; reagents obtained from AmershamPharmacia Biotech, Buckinghamshire, England) and the blot was exposed toX-ray film for up to 15 minutes.

The rabbit anti-human zcytoR17 MBP-6H was able to detect human zcytor17present in conditioned media as well as zcytoR17 purified protein as aband at 120 kDa under reducing conditions.

Example 26 Tissue Distribution of Mouse Zcytor17 in Tissue Panels UsingPCR

A panel of cDNAs from murine tissues was screened for mouse zcytor17expression using PCR. The panel was made in-house and contained 94marathon cDNA and cDNA samples from various normal and cancerous murinetissues and cell lines are shown in Table 6, below. The cDNAs came fromin-house libraries or marathon cDNAs from in-house RNA preps, ClontechRNA, or Invitrogen RNA. The mouse marathon cDNAs were made using themarathon-Ready™ kit (Clontech, Palo Alto, Calif.) and QC tested withmouse transferrin receptor primers ZC10,651 (SEQ ID NO:46) and ZC10,565(SEQ ID NO:47) and then diluted based on the intensity of thetransferrin band. To assure quality of the amplified library samples inthe panel, three tests for quality control (QC) were run: (1) To assessthe RNA quality used for the libraries, the in-house cDNAs were testedfor average insert size by PCR with vector oligos that were specific forthe vector sequences for an individual cDNA library; (2) Standardizationof the concentration of the cDNA in panel samples was achieved usingstandard PCR methods to amplify full length alpha tubulin or G3PDH cDNAusing a 5′ vector oligo: ZC14,063 (SEQ ID NO:48) and 3′ alpha tubulinspecific oligo primer ZC17,574 (SEQ ID NO:49) or 3′ G3PDH specific oligoprimer ZC17,600 (SEQ ID NO:50); and (3) a sample was sent to sequencingto check for possible ribosomal or mitochondrial DNA contamination. Thepanel was set up in a 96-well format that included a mouse genomic DNA(Clontech, Palo Alto, Calif.) positive control sample. Each wellcontained approximately 0.2-100 μg/μl of cDNA. The PCR was set up usingoligos ZC38,065 (SEQ ID NO:51) and ZC38,068 (SEQ ID NO:52), TaKaRa ExTaq™ (TAKARA Shuzo Co LTD, Biomedicals Group, Japan), and Rediload dye(Research Genetics, Inc., Huntsville, Ala.). The amplification wascarried out as follows: 1 cycle at 94° C. for 5 minutes; 5 cycles of 94°C. for 30 seconds, 68° C. for 30 seconds; 35 cycles of 94° C. for 30seconds, 56° C. for 30 seconds and 72° C. for 30 seconds, followed by 1cycle at 72° C. for 5 minutes. About 10 μl of the PCR reaction productwas subjected to standard Agarose gel electrophoresis using a 4% agarosegel. The correct predicted DNA fragment size was observed in brain,CD90+ cells, dendritic, embryo, MEWt#2, Tuvak-prostate cell line,salivary gland, skin and testis.

The DNA fragment for skin and testis were excised and purified using aGel Extraction Kit (Qiagen, Chatsworth, Calif.) according tomanufacturer's instructions. Fragments were confirmed by sequencing toshow that they were indeed mouse zcytor17. TABLE 6 Tissue/Cell line#samples Tissue/Cell line #samples 229 1 7F2 1 Adipocytes-Amplified 1aTC1.9 1 Brain 4 CCC4 1 CD90+ Amplified 1 OC10B 1 Dentritic 1 Embyro 1Heart 2 Kidney 3 Liver 2 Lung 2 MEWt#2 1 P388D1 1 Pancreas 1 Placenta 2Jakotay-Prostate Cell Line 1 Nelix-Prostate Cell Line 1 Paris-ProstateCell Line 1 Torres-Prostate Cell Line 1 Tuvak-Prostate Cell Line 1Salivary Gland 2 Skeletal Muscle 1 Skin 2 Small Intestine 1 SmoothMuscle 2 Spleen 2 Stomach 1 Testis 3 Thymus 1

Example 27 Human Zcytor17 Expression in Various Tissues Using Real-TimeQuantitative RT/PCR

A. Primers and Probes for Human Zcytor17, OSMRbeta and Zcytor17lig forConventional and Quantitative RT-PCR

Real-time quantitative RT-PCR using the ABI PRISM 7900 SequenceDetection System (PE Applied Biosystems, Inc., Foster City, Calif.) hasbeen previously described (See, Heid, C. A. et al., Genome Research6:986-994, 1996; Gibson, U. E. M. et al., Genome Research 6:995-1001,1996; Sundaresan, S. et al., Endocrinology 139:4756-4764, 1998). Thismethod incorporates use of a gene specific probe containing bothreporter and quencher fluorescent dyes. When the probe is intact thereporter dye emission is negated due to the close proximity of thequencher dye. During PCR extension using additional gene-specificforward and reverse primers, the probe is cleaved by 5′ nucleaseactivity of Taq polymerase which releases the reporter dye from theprobe resulting in an increase in fluorescent emission.

The primers and probes used for real-time quantitative RT-PCR analysesof human Zcytor17, OSMRbeta and Zcytor17ligand expression were designedusing the primer design software Primer Express™ (PE Applied Biosystems,Foster City, Calif.). Primers for human Zcytor17 were designed spanningan intron-exon junction to eliminate possible amplification of genomicDNA. The forward primer, ZC37,877 (SEQ ID NO:53) and the reverse primer,ZC37,876 (SEQ ID NO:54) were used in a PCR reaction at a 200 nMconcentration to synthesize a 73 bp product. The corresponding Zcytor17TaqMan® probe, designated ZC37,776 (SEQ ID NO:55) was synthesized andlabeled by PE Applied Biosystems and used in each PCR reaction at aconcentration of 200 nM. The ZC37,776 (SEQ ID NO:55) probe was labeledat the 5′ end with a reporter fluorescent dye (6-carboxy-fluorescein)(FAM) (PE Applied Biosystems) and at the 3′ end with a fluorescentquencher dye (6-carboxy-tetramethyl-rhodamine) (TAMRA) (EpochBiosciences, Bothell, Wash.).

Primers for human OSMRbeta were designed spanning an intron-exonjunction to eliminate possible amplification of genomic DNA. The forwardprimer, ZC43,891 (SEQ ID NO:137) and the reverse primer, ZC43,900 (SEQID NO:138) were used in a PCR reaction (below) at a 200 nMconcentration. The corresponding OSMRbeta TaqMan® probe, designatedZC43,896 (SEQ ID NO:139) was synthesized and labeled by PE AppliedBiosystems and used in each PCR reaction at a concentration of 200 nM.The ZC43,896 (SEQ ID NO:139) probe was labeled at the 5′end with areporter fluorescent dye (6-carboxy-fluorescein) (FAM) (PE AppliedBiosystems) and at the 3′ end with a non-fluorescent quencher dye(ECLIPSE) (Epoch Biosciences).

Primers for human Zcytor17ligand were designed spanning an intron-exonjunction to eliminate possible amplification of genomic DNA. The forwardprimer, ZC43,280 (SEQ ID NO:140) and the reverse primer, ZC43,281 (SEQID NO:141) were used in a PCR reaction (below) at about 200 nMconcentration. The corresponding Zcytor17ligand TaqMan® probe,designated ZC43,275 (SEQ ID NO:142) was synthesized and labeled by PEApplied Biosystems and used in each PCR reaction at a concentration of200 nM. The ZC43,275 (SEQ ID NO:142) probe was labeled at the 5′end witha reporter fluorescent dye (6-carboxy-fluorescein) (FAM) (PE AppliedBiosystems) and at the 3′ end with a non-fluorescent quencher dye(ECLIPSE) (Epoch Biosciences).

As a control to test the integrity and quality of RNA samples tested,all RNA samples were screened for either rRNA or GUS using primer andprobe sets either ordered from PE Applied Biosystems (rRNA kit) ordesigned in-house (GUS). The rRNA kit contained the forward primer (SEQID NO:56), the rRNA reverse primer (SEQ ID NO:57), and the rRNA TaqMan®probe (SEQ ID NO:58). The rRNA probe was labeled at the 5′end with areporter fluorescent dye VIC (PE Applied Biosystems) and at the 340 endwith the quencher fluorescent dye TAMRA (PE Applied Biosystems). The GUSprimers and probe were generated in-house and used in each PCR reactionat 200 nM and 100 nM, respectively. The forward primer was ZC40,574 (SEQID NO:143) and the reverse primer was ZC40,575 (SEQ ID NO:144). The GUSprobe ZC43,017 (SEQ ID NO:145) was labeled at the 5′end with a reporterfluorescent dye (Yakima-Yellow) (Epoch Biosciences) and at the 3′endwith a non-fluorescent quencher dye (ECLIPSE) (Epoch Biosciences). TherRNA and GUS results also serve as an endogenous control and allow forthe normalization of the Zcytor17 mRNA expression results seen in thetest samples.

For conventional non-quantitative RT-PCR, primers were designed usingthe primer design software Primer Express™ (PE Applied Biosystems,Foster City, Calif.). The human zcytor17 primers generate anapproximately 1000 base pair product and are as follows: forward primerZC28,917 (SEQ ID NO:83), and reverse primer ZC28,480 (SEQ ID NO:146).The human OSMRbeta primers generate a 202 base pair product and are asfollows: forward primer ZC41,653 (SEQ ID NO:147) and reverse primerZC41,655 (SEQ ID NO:148). The human Zcytor17ligand primers generate a305 base pair product and are as follows: forward primer ZC41,703 (SEQID NO:149) and reverse primer ZC41,704 (SEQ ID NO:150).

B. Primers and Probes for Murine Zcytor17, OSMRbeta and Zcytor17ligandfor Conventional and Quantitative RT-PCR

The primers and probes used for real-time quantitative RT-PCR analysesof murine Zcytor17, OSMRbeta and Zcytor17lig expression were designedusing the primer design software Primer Express™ (PE Applied Biosystems,Foster City, Calif.). Primers for murine Zcytor17 were designed spanningan intron-exon junction to eliminate possible amplification of genomicDNA. The forward primer, ZC43,272 (SEQ ID NO:151) and the reverseprimer, ZC43,273 (SEQ ID NO:152) were used in the PCR reactions (below)at 300 nM concentration. The corresponding Zcytor17 TaqMan® probe,designated ZC43,478 (SEQ ID NO:153) was synthesized and labeled by PEApplied Biosystems. The ZC43,478 (SEQ ID NO:153) probe was labeled atthe 5′end with a reporter fluorescent dye (6-carboxy-fluorescein) (FAM)(PE Applied Biosystems) and at the 3′ end with a quencher fluorescentdye (6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied Biosystems).The ZC43,478 (SEQ ID NO:153) probe was used in the PCR reactions at aconcentration of 100 nM.

Primers for murine Zcytor17ligand were designed spanning an intron-exonjunction to eliminate possible amplification of genomic DNA. The forwardprimer, ZC43,278 (SEQ ID NO:154) and the reverse primer, ZC43,279 (SEQID NO:155) were used in the PCR reactions at 500 nM concentration. Thecorresponding Zcytor17ligand TaqMan® probe, designated ZC43,276 (SEQ IDNO:156) was synthesized and labeled by PE Applied Biosystems. TheZC43,478 (SEQ ID NO:153) probe was labeled at the 5′end with a reporterfluorescent dye (6-carboxy-fluorescein) (FAM) (PE Applied Biosystems)and at the 3′ end with a non-fluorescent quencher dye (ECLIPSE) (EpochBiosciences). The ZC43,276 (SEQ ID NO:156) probe was used in the PCRreactions (below) at a concentration of 200 nM.

Primers for murine OSMRbeta were designed spanning an intron-exonjunction to eliminate possible amplification of genomic DNA. The forwardprimer, ZC43,045 (SEQ ID NO:157) and the reverse primer, ZC43,046 (SEQID NO:158) were used in the PCR reactions at a 300 nM concentration. Thecorresponding OSMRbeta TaqMan® probe, designated ZC43,141 (SEQ IDNO:159) was synthesized and labeled by Epoch Biosciences. The ZC43,141(SEQ ID NO:159) probe was labeled at the 5′end with a reporterfluorescent dye (6-carboxy-fluorescein) (FAM) (PE Applied Biosystems)and at the 3′ end with a non-fluorescent quencher dye (ECLIPSE) (EpochBiosciences). The ZC43,141 (SEQ ID NO:159) probe was used in the PCRreactions (below) at a concentration of 100 nM.

As a control to test the integrity and quality of RNA samples tested,all RNA samples were screened for either murine GUS or transferrinreceptor using primers and probes designed using the primer designprogram Primer Express™ (PE Applied Biosystems Inc., Foster City,Calif.). The murine GUS primers are as follows: forward primer, ZC43,004(SEQ ID NO:160), reverse primer, ZC43,005 (SEQ ID NO:161), and TaqMan®probe ZC43,018 (SEQ ID NO:162). The murine GUS probe ZC43,018 (SEQ IDNO:162) was labeled at the 5′end with a reporter fluorescent dyeYakima-Yellow (Epoch Biosciences) and at the 3′ end with thenon-fluorescent quencher dye ECLIPSE (Epoch Biosciences). The murine GUSprimers were used in the PCR reactions at 300 nM and the probe, ZC43,018(SEQ ID NO:162), was used at 100 nM. In some cases murine TransferrinReceptor was used instead of GUS as the endogenous control. Thetransferrin receptor forward primer, ZC40,269 (SEQ ID NO:163) and thereverse primer, ZC40,268 (SEQ ID NO:164) were used at 300 nM. Thetransferrin receptor probe, ZC40,298 (SEQ ID NO:165) was used in PCR at100 nM and was labeled at the 5′end with a reporter fluorescent dye VIC(PE Applied Biosystems) and at the 3′end with a fluorescent quencher dye(TAMRA) (PE Applied Biosystems). The murine GUS and transferrin receptorresults also serve as an endogenous control and allow for thenormalization of the Zcytor17, OSMRbeta and Zcytor17ligand mRNAexpression results seen in the test samples.

For conventional semi-quantitative RT-PCR, primers were designed usingthe primer design software Primer Express™ (PE Applied Biosystems). Themurine Zcytor17 primers generate a 276 base pair product and are asfollows: forward primer ZC43,140 (SEQ ID NO:166), and reverse primerZC43,139 (SEQ ID NO:167). The murine OSMRbeta primers generate a 575base pair product and are as follows: forward primer ZC41,608 (SEQ IDNO:168) and reverse primer ZC41,609 (SEQ ID NO:169). The murineZcytor17ligand primers generate a 657 bp product and are as follows:forward primer ZC41,502 (SEQ ID NO:170) and reverse primer ZC41,500 (SEQID NO:171).

C. Protocols for Realtime Quantitative RT-PCR and ConventionalSemi-quantitative RT-PCR

Relative levels of Zcytor17, OSMRbeta and Zcytor17ligand mRNA weredetermined by analyzing total RNA samples using the one-step RT-PCRmethod (PE Applied Biosystems). Total RNA from Zcytor17- andOSMRbeta-transfected BAF cells (human) or BHK cells (murine) wasisolated by standard methods and used to generate a standard curve usedfor quantitation of Zcytor17 and OSMRbeta. The curve consisted of10-fold serial dilutions ranging from 100-0.01 ng/μl with each standardcurve point analyzed in triplicate. Similarly, for Zcytor17ligand,activated CD4+ T cell RNA (previously shown to make Zcytor17ligand) wasused to generate a standard curve in the same 100-0.01 ng/μl range.Total RNA from human or murine cells was analyzed in triplicate foreither human or murine Zcytor17, OSMRbeta and Zcytor17ligand transcriptlevels and for one of the following endogenous control genes: rRNA, GUSor transferrin receptor. In a total volume of 10 μl, each RNA sample wassubjected to a One-Step RT-PCR reaction containing: approximately 50-100ng of total RNA in a preformulated 2× master mix containing an internalcontrol dye (ROX)(carboxy-x-rhodamine) and Thermo-Start® DNA Polymerase(Abgene, Surrey, UK); appropriate primers for the gene of interest (seeparts A and B of current example); the appropriate probe (see parts Aand B for concentration); Superscript reverse transcriptase (50 U/μl)(PE Applied Biosystems), and an appropriate volume of RNase-free water.PCR thermal cycling conditions were as follows: an initial reversetranscription (RT) step of one cycle at 48° C. for 30 minutes; followedby a Thermo-Start® enzyme activation step of one cycle at 95° C. for 10minutes; followed by 40 cycles of amplification at 95° C. for 15 secondsand 60° C. for 1 minute. Relative Zcytor17, OSMRbeta and Zcytor17ligandRNA levels were determined by using the Standard Curve Method asdescribed by the manufacturer, PE Biosystems (User Bulletin #2: ABIPrism 7700 Sequence Detection System, Relative Quantitation of GeneExpression, Dec. 11, 1997). The rRNA, GUS or Transferrin Receptormeasurements were used to normalize the levels of the gene of interest.

The semi-quantitative RT-PCR reactions used the ‘Superscript One-StepRT-PCR System with Platinum Taq’ (Invitrogen, Carlsbad, Calif.). Each 25μl reaction consisted of the following: 12.5 μl of 2× Reaction Buffer,0.5 μl (20 pmol/μl) of forward primer, 0.5 μl (20 pmol/μl) of reverseprimer, 0.4 μl RT/Taq polymerase mix, 5.0 μl of Rediload Gel LoadingBuffer (Invitrogen), 5.1 μl RNase-free water, and 10 μl total RNA (100ng/μl). The amplification was carried out as follows: one cycle at 45°C. for 30 minutes followed by 35-38 cycles of 94° C., 20 seconds;Variable annealing temp (See Table 7 below), 20 seconds; 72° C., 45seconds; then ended with a final extension at 72° C. for 5 minutes.Eight to ten microliters of the PCR reaction product was subjected tostandard agarose gel electrophoresis using a 2% agarose gel. TABLE 7Murine Zcytor17 58° C. anneal temp Murine OSMRbeta 60° C. anneal tempMurine 52° C. anneal temp Zcytor17ligand Human Zcytor17 55° C. annealtemp Human OSMRbeta 59° C. anneal temp Human 59° C. anneal tempZcytor17ligandD. Isolation of RNA from Human and Murine PBMC Subsets and Cell Lines

Blood was drawn from several anonymous donors and peripheral bloodmononuclear cells (PBMC) isolated using standard Ficoll gradientmethodology. Monocytes were then isolated using the Monocyte IsolationKit and the Magnetic Cell Separation System (Miltenyi Biotec, Auburn,Calif.). The monocytes were then plated onto ultra-low adherence 24-wellplates in endotoxin-free media. They were either unstimulated or treatedwith recombinant human IFNg (R&D Systems Inc.) at 10 ng/ml. Cells werecollected at 24 and 48 hours. In similar manner, CD4+ and CD8+ T cellswere isolated from PBMC's using the anti-CD4 or anti-CD8 magnetic beadsfrom Miltenyi Biotec. Cells were then activated for 4 or 16 hours intissue culture plates coated with 0.5 μg/ml anti-CD3 antibodies in mediacontaining 5 μg/ml anti-CD28 antibodies. NK cells were also isolatedfrom PBMC's using Miltenyi's anti-CD56 coated magnetic beads. Some ofthe NK cells were collected at time zero for RNA and the others wereplated in media containing Phorbol Myristate Acetate (PMA) (5 ng/ml) andionomycin (0.5 μg/ml) for 24 hours. Additionally, several humanmonocyte-like cell lines, U937, THP-1 and HL-60, were collected ineither their resting or activated states. U937 cells were activatedovernight with PMA (10 ng/ml). HL-60's were activated overnight with PMA(10 ng/ml) or for 72 and 96 hours with IFNg (10 ng/ml) to drive themdown a monocytic pathway. THP-1 cells were activated overnight with acombination of LPS (10 ng/ml) and IFNg (10 ng/ml). RNA was prepared fromall primary cells using the RNeasy Midiprep™ Kit (Qiagen, Valencia,Calif.) as per manufacturer's instructions. Carryover DNA was removedusing the DNA-Free™ kit (Ambion, Inc., Austin, Tex.). RNA concentrationwas determined using standard spectrophotometry and RNA qualitydetermined using the Bioanalyzer 2100 (Agilent Technologies, Palo Alto,Calif.).

Murine T Cell RNA was collected using a variety of methods well-known inthe art. Primary splenic CD4+ and CD8+ T cells were isolated from thespleens of C57B1/6 mice using antibody-coated magnetic beads and theMagnetic Cell Separation System from Miltenyi Biotec. The CD4+ and CD8+T cells were then activated by culturing the cells in 24-well platescoated with anti-CD3 antibodies (500 ng/ml) in media containinganti-CD28 antibodies at 5 μg/ml. Cells were harvested for RNA at 0, 4and 16 hours. Similarly, CD4+ T cells were isolated and then skewedtowards a Th1 or Th2 phenotype using the following protocol. SinceC57B1/6 T cells are already skewed in the Th1 direction, all that wasrequired was to activate for 6 hours with 0.5 μg/ml PMA and long/mlionomycin. ‘Th2’ skewing was obtained by plating naïve CD4+ T cells with2.5 μg/ml anti-CD28, 10 ng/ml mIL-2 (R&D Systems Inc.) and 25 ng/mlmIL-4 (R&D Systems) into plates coated with 0.5 μg/ml anti-CD3. After 2days in culture, cells were resuspended in media containing 10 ng/mlmIL-2 (R&D Systems) and 25 ng/ml mIL-4. Cells were cultured for anadditional three days then activated with PMA and ionomycin for 6 hours.

One additional set of Th1 and Th2 skewed T cells was derived using the TCell Receptor Transgenic DO11.10 T cell line. All cells were plated intoanti-CD3 and anti-CD28 coated plates. The ‘Th1’ cells were plated inmedia containing mIL-12 (1 ng/ml) and anti-IL-4 (10 μg/ml). The ‘Th2’cells were plated in media containing mIL-4 (10 ng/ml) and anti-IFNg (10μg/ml). After 24 hours, all cultures were given mIL-2 (10 ng/ml). Aftertwo more days, the media on the cells was changed and new mediacontaining the aforementioned cytokines was added and cells werecultured an additional 4 days before being harvested.

All of the murine T cell RNA was prepared using the RNeasy Midiprep™ Kit(Qiagen) and contaminating DNA was removed using the DNA-free™ kit fromAmbion.

E. Isolation of RNA from the Murine Models of Pancreatitis and IrritableBowel Disease

To induce a condition similar to human Irritable Bowel Disease (IBD),the hybrid mouse strain C57B16/129S6F1 was used. Mice were divided into4 groups with an average size of six mice per group. Group 1 was givenno Dextran Sulfate Sodium (DSS) and was sacrificed on day 14. Group 2received 2% DSS for two days prior to being sacrificed. Group 3 received2% DSS for seven days prior to sacrifice. Group 4 received 2% DSS forseven days then allowed to recover for seven days and was sacrificed onday 14. On the day of sacrifice, the distal colon sections were removedand placed in RNAlater™ (Ambion). The colon sections were homogenizedusing standard techniques and RNA was isolated using the RNeasyMidiprep™ Kit (Qiagen). Contaminating DNA was removed by DNA-free™(Ambion) treatment as per manufacturer's instructions.

In a different study, acute pancreatitis was induced in male CD-1 miceby caerulein injection. Mice were divided into three groups (n=8mice/group). Group 1 animals were given seven i.p. injections (1injection per hour) with Vehicle (saline), and then sacrificed at 12 and24 hours following the first injection. Groups 2 and 3 were given seveni.p. injections of caerulein (Sigma) (Catalog#C-9026) at a dose of 50μg/kg/hr for six hours (1 injection per hour). Group 2 was sacrificed at12 hrs after the first injection and Group 3 was sacrificed at 24 hrsfollowing the first injection. Pancreases were removed at the time ofsacrifice and snap frozen for RNA isolation. Tissues were homogenizedand RNA was isolated using the Qiagen RNeasy Midiprep™ Kit.

In yet another study, murine Zcytor17ligand transgenic mice weregenerated and observed for phenotypic changes (see Example 41).Piloerection and hair loss was observed in many of the transgenic mice.Four transgenic mice were sacrificed and skin samples from both normaland hairless areas were removed and snap frozen for later RNA isolation.Skin sections from two non-transgenic control mice were collected aswell. Skin samples were homogenized and then digested with Proteinase K(Qiagen) (Catalog# 19133) for 20 minutes at 60° C. RNA was then isolatedusing the Qiagen RNeasy Midiprep™ Kit following manufacturer'sinstructions. Carryover DNA was removed using DNA-free™ kit from Ambion.

F. Results of Quantitative and Semi-Quantitative RT-PCR for HumanZcytor17, OSMRbeta and Zcytor17ligand

Zcytor17 and OSMRbeta expression was examined by quantitative RT-PCR infour sets of primary human monocytes that were either in their restingstate or activated with IFNg for 24 or 48 hours. Zcytor17 expression wasbelow detection in the unstimulated cells but increased dramaticallyafter the 24-hour activation with IFNg, and was the highest after 48hours of activation. In all cases OSMRbeta was below detection.Zcytor17ligand was not tested in these samples.

In the primary T cells, Zcytor17 was below detection in both the restingCD4+ and CD8+ subsets. After a four-hour activation, however, expressionof Zcytor17 went up in both subsets and then decreased to a slightlylower level at the 16 hour time point. OSMRbeta was below detection inthese samples. Zcytor17ligand expression was examined usingsemi-quantitative RT-PCR. No expression was detected in the unstimulatedCD4+ and CD8+ T cells. However, after the four hour activation, highlevels of Zcytor17ligand were detected. This level dropped somewhat atthe 16 hour time point.

Expression of Zcytor17 was not examined in NK cells. OSMRb was belowdetection in these samples. Zcytor17ligand expression was belowdetection in the resting NK cells, however there was a faint signalgenerated by the activated NK cells suggesting that these cells may makeZcytor17ligand under certain conditions.

In the human monocyte-like cell lines, U937, THP-1 and HL-60, OSMRbetaexpression was below detection in all of the resting and activatedsamples except for activated THP-1 samples where a faint signal wasdetected. Zcytor17 expression was high in both the U937 and THP-1resting cell lines and showed a strong upregulation followingactivation. Expression in U937's was the highest of any cell type. Inthe HL-60's, Zcytor17 was expressed at moderate levels in theunstimulated cells and decreased upon stimulation with PMA. However, theexpression of Zcytor17 was dramatically upregulated in the HL-60's whenstimulated with IFNg for 72 and 96 hours. All of the human expressiondata is summarized in Table 8 below. TABLE 8 Primary Human ActivationMonocytes Status Zcytor17 OSMRbeta Zcytor17Ligand Human Unstim − −Monocytes Human Act. 24 hr + − Monocytes IFNg Human Act. 48 hr ++ −Monocytes IFNg Human CD4+ Unstim − − − Human CD4+ Act 4 hr ++ − ++ HumanCD4+ Act. 16 hr + − + Human CD8+ Unstim − − − Human CD8+ Act 4 hr ++ −++ Human CD8+ Act. 16 hr + − + Human NK Unstim − − Cells Human NK Act 24hr − + Cells U937 Unstim ++ − − U937 Act. 16 hr +++ − − THP-1 Unstim ++− − THP-1 Act. 16 hr +++ + − HL-60 Unstim ++ − − HL-60 Act. 16 hr + − −PMA HL-60 Act. 72 hr +++ − − IFNg HL-60 Act. 96 hr +++ − − IFNgG. Results of Quantitative and Semi-Quantitative RT-PCR for MurineZcytor17, OSMRbeta and Zcytor17ligand

Murine Zcytor17, OSMRbeta and Zcytor17ligand expression levels wereexamined in several murine T cells populations and the results aresummarized in Table 9 below. Murine Zcytor17 expression was tested bysemi-quantitative RT-PCR and shown to be at low levels on both restingand activated primary CD4+ T cells. Expression of Zcytor17 was detectedon resting CD8+ T cells and then seemed to drop upon activation withanti-CD3 and anti-CD28 antibodies at both the 4- and 16-hour timepoints. OSMRbeta expression was measured by quantitative RT-PCR andshown to be expressed in resting and activated CD4+ and CD8+ T cells.The expression of OSMRbeta went up after a 4-hour activation and thenreturned to the unstimulated levels by 16 hours in both the CD4+ andCD8+ T cells. Zcytor17ligand was detected by quantitative RT-PCR andshown to be expressed at very low levels in unstimulated CD4+ T cells.However, following a 4-hour activation, Zcytor17ligand expression wasdramatically upregulated and then dropped slightly by the 16-hour timepoint. In CD8+ T cells, no Zcytor17ligand was detected in theunstimulated cells. There was some Zcytor17ligand expression at the4-hour time point, but by 16 hours expression levels had dropped backbelow detection.

In the DO11.10 T cells, Zcytor17 expression was detected in the naïveand Th2 skewed cells, but not in the Th1 skewed cells. OSMRbetaexpression was at low levels in the naïve DO11.10 cells. There was adramatic increase in OSMRbeta expression levels in the Th1 skewed cellsand a moderate increase of expression in the Th2-skewed cells. TheZcytor17ligand expression in these cells was shown to be predominantlyby the Th2 skewed subset. Low levels were detected in the Th1 subset andno expression was detected in the naïve cells. These results aresummarized in the Table 9 below.

In the primary CD4+ T cells that were skewed in either the Th1 or Th2direction, Zcyto17 wasn't examined. OSMRbeta expression was detected inall three samples with the highest levels found in the Th2 sample.Similar to the DO11.10 results, Zcytor17ligand expression was detectedat high levels in the Th2 skewed subset, with a small amount detected inthe Th1 subset and levels were below detection in the unstimulatedcells. These results are summarized in the Table 9 below. TABLE 9 MurineT Cells Zcytor17 OSMRbeta Zcytor17ligand CD4+ T Cells Unstimulated + ++/− CD4+ T Cells 4 hr Activation + ++ ++ CD4+ T Cells 16 hr + + +Activation CD8+ T Cells Unstimulated + + − CD8+ T Cells 4 hr Activation+/− ++ + CD8+ T Cells 16 hr − + − Activation DO11.10 Naïve + + − DO11.10Th1 − +++ + DO11.10 Th2 + ++ ++ CD4+ T Cells Unstimulated ++ − CD4+ TCells - Th1 Skewed +++ + CD4+ T Cells - Th2 Skewed ++ +++

In the Zcytor17ligand transgenic skin samples, Zcytor17, OSMRbeta andZcytor17ligand expression levels were determined using quantitativeRT-PCR. Zcytor17 was shown to be present in all samples at roughlyequivalent levels. There were dramatically higher levels of OSMRbetaexpression in the non-transgenic control animals than the transgenicsamples. Zcytor17ligand expression was below detection in thenon-transgenic control animals with moderate to high levels ofexpression in the transgenic animals. The results are summarized inTable 10 below. TABLE 10 Murine Zcytor17ligand Skin Transgenic SkinPhenotype Zcytor17 OSMRbeta Zcytor17ligand Wild Type Mouse Normal + +++− Wild Type Mouse Normal + +++ − Transgenic #1 Normal + + + Transgenic#1 Hair Loss + + + Transgenic #2 Normal + + + Transgenic #2 HairLoss + + + Transgenic #3 Normal + + + Transgenic #3 Hair Loss + + +Transgenic #4 Normal + + +++ Transgenic #4 Hair Loss + + +++

In a different experiment, Zcytor17, OSMRbeta and Zcytor17ligandexpression levels were measured by quantitative RT-PCR in the pancreasesof mice subjected to acute pancreatitis. Zcytor17 expression was belowdetection in all of the samples. OSMRbeta expression was seen at lowlevels in the normal control samples (Group 1), but showed a strongupregulation at the 12-hour time point (Group 2) and slightly lowerlevels at the 24-hour time point (Group 3). Zcytor17ligand expressionwas below detection in the control animals, but showed high levels inboth of the caerulein injected groups. The data is summarized in Table11 below. TABLE 11 Pancreatitis Model Description Zcytor17 OSMRbetaZcytor17ligand Group 1 Normal Control − + − Group 2 12 hr Post − +++ ++Injection Group 3 24 hr Post − ++ ++ Injection

In another experiment, Zcytor17 and OSMRbeta expression levels wereexamined in the distal colons of mice subjected to DSS treatment. Inthis murine model of Inflammatory Bowel Disease, expression levels ofboth genes were determined by quantitative RT-PCR and are summarized inTable 12 below. Zcytor17 expression levels increased with the severityof the disease, with low levels of expression in the Group 1 normalanimals and increasing amounts seen Groups 2 and 3. In the Group 4animals, the Zcytor17 levels had returned to more normal levels. UnlikeZcytor17 expression, OSMRbeta levels were the highest in the controlanimals and levels actually decreased in all three DSS treated groups.TABLE 12 IBD Model Description SAC Day Zcytor17 OSMRbeta Group 1 NormalControl 14 + ++ Group 2 DSS-Treated 2 days 2 ++ + Group 3 DSS-Treated 7days 7 +++ + Group 4 DSS-Treated 7 days 14 + +

Example 28 Human Zcytor17lig Tissue Distribution Expression Based onRT-PCR Analysis of Multiple Tissue First-Strand cDNAs

Gene expression of the zcytor17lig was examined using commerciallyavailable normalized multiple tissue first-strand cDNA panels (OriGeneTechnologies, Inc. Rockville, Md.; BD Biosciences Clontech, Palo Alto,Calif.). These included the OriGene “Human Tissue Rapid-Scan™ Panel”(Cat. #CHSCA-101, containing 22 different tissues, bone marrow, andplasma blood leucocytes) and the BD Biosciences Clontech “Human BloodFractions MTC™ Panel” (Cat. #K1428-1, containing 9 different bloodfractions).

PCR reactions were set up using the zcytor17lig specific oligo primersZC41,458 (SEQ ID NO:60), and ZC41,457 (SEQ ID NO:61), which yield a 139bp product, and ZC41,459 (SEQ ID NO: 62), and ZC41,460 (SEQ ID NO:63),which yield a 92 bp product, Qiagen HotStarTaq DNA polymerase and buffer(Qiagen, Inc., Valencia, Calif.), dH₂H, and RediLoad™ dye (ResearchGenetics, Inc., Huntville, Ala.). The PCR cycler conditions were asfollows: an initial 1 cycle 15 minute denaturation at 95° C., 35 cyclesof a 45 second denaturation at 95° C., 1 minute annealing at 53° C. or56° C. and 1 minute and 15 seconds extension at 72° C., followed by afinal 1 cycle extension of 7 minutes at 72° C. The reactions wereseparated by electrophoresis on a 2% agarose gel (EM Science, Gibbstown,N.J.) and visualized by staining with ethidium bromide.

A DNA fragment of the correct size was observed in the following humanadult tissues using the OriGene “Human Tissue Rapid-Scan™ Panel”:testis, plasma blood leucocytes (PBL), and bone marrow.

A DNA fragment of the correct size was observed in the following humanblood fractions using the BD Biosciences Clontech “Human Blood FractionsMTC™ Panel”: activated mononuclear cells (B- & T-cells and monocytes),activated CD8+ cells (T-suppressor/cytotoxic), activated CD4+ cells(T-helper/inducer) and faintly in resting CD8+ cells.

Example 29 Cloning the Human Oncostatin M Receptor

The OncostatinM beta receptor (OSMRbeta) is a type I cytokine receptorwith structural similarity to IL12R-B2. ZcytoR17 has structuralsimilarity to IL12R-B1. The OSMRbeta and zcytor17 were tested to seewhether they could interact as subunits in a cytokine signaling complex,and whether together they could act as a signaling receptor, or solublereceptor antagonist, for zcytor17lig.

To isolate OSMRbeta, oligonucleotide PCR primers ZC39982 (SEQ ID NO:64)and ZC39983 (SEQ ID NO:65) were designed to amplify the full lengthcoding region of the human OncostatinM beta cDNA sequence (SEQ ID NO:6)(Genbank Accession No. U60805; Mosley B, JBC Volume 271, Number 50,Issue of Dec. 20, 1996 pp. 32635-32643).

PCR reactions were run on an array of cDNA library templates using arobust polymerase, Advantage II (Clonetech, PaloAlto, Calif.), in orderto identify a source of the cDNA. The template DNA used was fromamplified cDNA plasmid libraries each containing 5 million independentcDNA clones. Reactions were assembled as per manufacturer's instructionsusing 400 fmol/μl of each oligonucleotide and 2-20 ng/μl purifiedplasmid library DNA as template. The cDNA libraries were derived fromthe following human tissues and cell lines: fetal brain, prostate smoothmuscle, bone marrow, RPM11588, thyroid, WI-38, testis, stimulatedperipheral blood mononuclear cells, stimulated CD3+ cells, THP-1,activated tonsil, HACAT and fetal liver. Reactions were performed on athermocycler machine using the following conditions: 30 cycles of 95° C.for 20 seconds, 68° C. for 3 minutes. At the conclusion of 30 cycles anadditional single extension cycle of 8 minutes at 68° C. was run. PCRproducts were visualized by TAE agarose, gel electrophoresis in thepresence of ethidium bromide followed by UV illumination. The mostabundant product was found to be from a prostate smooth muscle cDNAlibrary. The PCR reaction using prostate smooth muscle template andoligonucleotides ZC39982 (SEQ ID NO:64) and ZC39983 (SEQ ID NO:65) wasrepeated using a less robust but higher fidelity thermostable DNApolymerase “turboPFu”, (Stratagene, La Jolla, Calif.). Thirtyamplification cycles were run with the following conditions: denaturingat 94° C., 30 seconds, annealing at 63° C. 45 seconds, extension at 72°C. 3.5 minutes. A single band product was gel purified on a 0.8% TAE,agarose gel.

This DNA was then amplified again using primers ZC39980 (SEQ ID NO:66)and ZC39981 (SEQ ID NO:67) designed to include restriction enzymerecognition sequences to allow the cloning of this cDNA into a mammalianexpression vector.

The PCR reaction was performed using “TurboPfu” and the purified PCRproduct for 15 cycles of: 95° C. 1 minute, 64° C. 1 minute 20 seconds,72° C. 4.5 minutes. The PCR reaction was then digested with EcoR1 andXho1 (Invitrogen, Carlsbad, Calif.) and gel purified as described above.A mammalian expression vector, pZ7NX, was prepared by digesting withEcoR1 and Xho1 and the PCR product was ligated to this vector andelectroporated into E. coli DH10b cells. Several bacterial colonies wereisolated and sequenced. One clone was correct with the exception of asingle non-conservative mutation. In order to change this base to matchthe expected sequence, an oligonucleotide spanning mutation and aneighboring Pst1 restriction site was used in a PCR reaction with“TurboPfu” using the pZP7Nx-h. OncostatinM R plasmid previouslysequenced as a template. The PCR amplified DNA was digested with Pst1and Xho1 and cloned back into the pZP7Nx-h OncostatinM R plasmid inplace of the Pst1/Xho1 fragment containing the offending mutation. Thisnew plasmid was sequenced over the recently amplified Pst1 to Xho1region to confirm the correction and make sure no other errors werecreated in the amplification process. This analysis confirmed sequencethat matched the expected sequence over the coding region. The sequenceis shown in SEQ ID NO:6, and corresponding amino acid sequence shown inSEQ ID NO:7.

Example 30 Constructs for Generating a Human Zcytor17/OncostatinMReceptor (OSMRbeta) Heterodimer

A system for construction, expression and purification of such solubleheterodimeric receptors is known in the art, and has been adapted to thereceptor pair, human oncostatin M receptor (OSMRbeta) and humanzcytor17. For this construct, the polynucleotide for the solublereceptor for OSMRbeta is shown in SEQ ID NO:68 and correspondingpolypeptide is shown in SEQ ID NO:69; and the polynucleotide for thesoluble receptor for human zcytor17 is shown in SEQ ID NO:70 andcorresponding polypeptide is shown in SEQ ID NO:71.

To construct a cell line expressing a secreted soluble hzcytor17/humanOSMRbeta heterodimer, a construct was made so that the resultingheterodimeric soluble receptor comprises the extracellular domain ofhuman OSMRbeta fused to the heavy chain of IgG gamma1 (Fc4) (SEQ IDNO:37) with a Glu-Glu tag (SEQ ID NO:35) at the C-terminus; while theextracellular domain of zcytoR17 is fused to Fc4 (SEQ ID NO:37) with aHis tag (SEQ ID NO:72) at the C-terminus. For both of the hzcytor17 andhuman OSMRbeta arms of the heterodimer a Gly-Ser spacer of 12 aminoacids (SEQ ID NO:73) was engineered between the extracellular portion ofthe receptor and the N-terminus of Fc4.

A. Construction of Human Soluble OSMRbeta/Fc4-CEE

For construction of the human soluble OSMRbeta/Fc4-CEE portion of theheterodimer the extracellular portion of human OSMRbeta was isolatedusing PCR with oligos ZC14063 (SEQ ID NO:48) and ZC41557 (SEQ ID NO:74)under PCR reaction conditions as follows: 30 cycles of 95° C. for 60sec., 57° C. for 30 sec., and 72° C. for 100 sec.; and 72° C. for 7 min.PCR products were purified using QIAquick PCR Purification Kit (Qiagen),digested with EcoR1 and BglII (Boerhinger-Mannheim), separated by gelelectrophoresis and purified using a QIAquick gel extraction kit(Qiagen).

The expression cassette, plasmid backbone and Fc4-GluGlu tag portion ofthe chimera were contained within a previously made in house plasmidvector. The plasmid vector was digested with EcoR1 and BamH1(Boerhinger-Mannheim), separated by gel electrophoresis and purifiedusing a QIAquick gel extraction kit (Qiagen). The digested and purifiedfragments of human OSMRbeta and Fc4-cEE containing plasmid were ligatedtogether using T4 DNA Ligase (Life Technologies, Bethesda, Md.) usingstandard ligation methods. Minipreps of the resulting ligation werescreened for an EcoRI/Sma1 insert of the correct size (772 bp) for thesoluble OSMRbeta and positive minipreps were sequenced to confirmaccuracy of the PCR reaction. This new plasmid construction is termedpZP9-ONCOMR-Fc4CEE.

B. Construction of Human Soluble Zcytor17/Fc4-CHIS

For construction of the hzcytor17/Fc4-CHIS portion of the heterodimer,the extracellular portion of human zcytor17 was isolated by digestion ofa plasmid previously containing Zcytor17-Fc4 soluble receptor. Theplasmid was first digested with Sal1 (New England Biolabs, Beverly,Mass.) after which the reaction was serially phenol chloroform extractedand ethanol precipitated. The digested DNA was then treated with T4 DNAPolymerase (Boerhinger-Mannheim), to fill in the 5′ overhangs created bythe Sal1 digestion, leaving the DNA ends blunt, after which the reactionwas serially phenol chloroform extracted and ethanol precipitated. Theblunted DNA was then further digested with BglII to cut at the 3′ end.),separated by gel electrophoresis and purified using a QIAquick gelextraction kit (Qiagen) as per manufacturer's instruction. The resultingDNA fragment containing the sequence coding for the extracellular domainof zcytoR17 was ligated into an Fc4-CHIS tag containing mammalianexpression vector prepared as follows.

The expression cassette, plasmid backbone and Fc4-CHIS tag portion ofthe chimera were contained within a previously made in house plasmidvector. This plasmid vector was digested with EcoR1(Boerhinger-Mannheim) after which the reaction was serially phenolchloroform extracted and ethanol precipitated. The digested DNA was thentreated with T4 DNA Polymerase (Boerhinger-Mannheim), to fill in the 5′overhangs created by the EcoRI digestion, leaving the DNA ends blunt,after which the reaction was serially phenol chloroform extracted andethanol precipitated. The blunted DNA was then further digested withBamH1 (Boerhinger-Mannheim) to cut at the 3′ end, separated by gelelectrophoresis and purified using a QIAquick gel extraction kit(Qiagen). The digested and purified fragments of human zcytor17 andFc4-CHIS containing plasmid were ligated together using T4 DNA Ligase(Life Technologies, Bethesda, Md.) using standard ligation methods.

Minipreps of the resulting ligation were screened by PCR using thezcytor17 specific sense primer ZC29180 (SEQ ID NO:22) and the Fc4specific antisense primer ZC29232 (SEQ ID NO:75) with the following PCRreaction conditions: 30 cycles of 94° C. for 60 sec., 68° C. for 150sec; and 72° C. for 7 min. An expected product size of 848 bp confirmedthe correct assembly of the plasmid termed pZEM228 hzcytor17/Fc4HIS.

A second zcytor17-Fc4 construction was created for use in generatinghomodimer protein from COS cells. Briefly the coding region for the fullfusion protein was isolated by digestion of a plasmid previouslycontaining Zcytor17-Fc4 soluble receptor with Sal1(Boerhinger-Mannheim). The reaction was serially phenol chloroformextracted and ethanol precipitated. The digested DNA was then treatedwith T4 DNA Polymerase (Boerhinger-Mannheim), to fill in the 5′overhangs created by the EcoRI digestion, leaving the DNA ends blunt,after which the reaction was serially phenol chloroform extracted andethanol precipitated. The blunted DNA was then further digested withNot1 (Boerhinger-Mannheim) to cut at the 3′ end, separated by gelelectrophoresis and purified using a QIAquick gel extraction kit(Qiagen). A mammalian expression vector containing a CMV drivenexpression cassette was digested to generate compatible ends and the 2fragments were ligated together. Minipreps of the resulting ligationwere screened by PCR using the vector specific sense primer ZC14063 (SEQID NO:48) and the zcytor17 specific antisense primer ZC27899 (SEQ IDNO:19) with the following PCR reaction conditions: 30 cycles of 94° C.for 30 sec., 64° C. for 30 sec; 70° C. for 90 sec; and 72° C. for 7 min.An expected product size of approximately 1000 bp confirmed the correctassembly of the plasmid termed pZP7NX-hzcytor17-Fc4. This plasmid wassubsequently transfected into COS cells using Lipofectamine (Gibco/BRL),as per manufacturer's instructions. The cells were conditioned for 60hours in DMEM+5% FBS (Gibco/BRL) after which the protein was purifiedover a protein G-sepharose 4B chromatography column and made availablefor in vitro bioassays, for example, such as those described herein.

C. Generating a Human Zcytor17/OncostatinM Receptor (OSMRbeta)

About 16 μg each of the pZP9-ONCOMR-Fc4CEE and pZEM228 hzcytor17/Fc4HISwere co-transfected into BHK-570 (ATCC No. CRL-10314) cells usinglipofectamine (Gibco/BRL), as per manufacturer's instructions. Thetransfected cells were selected for 10 days in DMEM+5% FBS (Gibco/BRL)containing 0.5 mg/ml G418 (Gibco/BRL) and 250 nM methyltrexate(MTX)(Sigma, St. Louis, Mo.) for 10 days.

The resulting pool of doubly-selected cells was used to generate theheterodimeric protein. Three cell Factories (Nunc, Denmark) of this poolwere used to generate 10 L of serum free conditioned medium. Thisconditioned media was passed over a 1 ml protein-A column and eluted in(10) 750 microliter fractions. Four of these fractions found to have thehighest concentration were pooled and dialyzed (10 kD MW cutoff) againstPBS. The desired heterodimeric soluble zcytor17/OSMRbeta protein complexwas isolated from other media components by passing the pool over aNickel column and washing the column with various concentrations ofImidazole. The soluble zcytor17/OSMRbeta protein eluted at intermediateconcentrations of Imidazole, while hzcytor17/Fc4HIS homodimer eluted athigher concentrations of Imidazole.

Example 31 Tissue Distribution of Human Zcytor17 in Tissue Panels UsingNorthern Blot and PCR

A. Human Zcytor17 Tissue Distribution Using Northern Blot

Human Multiple Tissue Northern Blots (Human 12-lane MTN Blot I and II,and Human Immune System MTN Blot II; Human Endocrine MTN, Human FetalMTN Blot II, Human Multiple Tissue Array) (Clontech) as well as in houseblots containing various tissues were probed to determine the tissuedistribution of human zcytor17 expression. The in-house prepared blotsincluded the following tissue and cell line mRNA: SK-Hep-1 cells, THP1cells, Adrenal gland (Clontech); Kidney (Clontech), Liver (Clontech andInvitrogen); Spinal cord (Clontech), Testis (Clontech), Human CD4+T-cells, Human CD8+ T-cells, Human CD19+ T-cells, human mixed lymphocytereaction (MLR), THP1 cell line (ATCC No. TIB-202), U937 cell line,p388D1 mouse lymphoblast cell line (ATCC No. CCL-46) with or withoutstimulation by Ionomycin; and WI-38 human embryonic lung cell line (ATCCNo. CRL-2221) with or without stimulation by Ionomycin.

An approximately 500 bp PCR derived probe for zcytor17 (SE ID NO:4) wasamplified using oligonucleotides ZC28,575 (SEQ ID NO:77) and ZC27,899(SEQ ID NO:19) as primers. The PCR amplification was carried out asfollows: 30 cycles of 94° C. for 1 minute, 65° C. for 1 minute, and 72°C. for 1 minute; followed by 1 cycle at 72° C. for 7 minutes. The PCRproduct was visualized by agarose gel electrophoresis and theapproximately 500 bp PCR product was gel purified as described herein.The probe was radioactively labeled using the PRIME IT II™ Random PrimerLabeling Kit (Stratagene) according to the manufacturer's instructions.The probe was purified using a NUCTRAP™ push column (Stratagene).EXPRESSHYB™ (Clontech) solution was used for the prehybridization and asa hybridizing solution for the Northern blots. Prehybridization wascarried out at 68° C. for 2 hours. Hybridization took place overnight at68° C. with about 1.5×10⁶ cpm/ml of labeled probe. The blots were washedthree times at room temperature in 2×SSC, 0.05% SDS, followed by 1 washfor 10 minutes in 2×SSC, 0.1% SDS at 50° C. Several faint bands wereseen after several days exposure. An approximately 9 kb transcript wasseen in trachea, skeletal muscle and thymus; an approximately 2 kbtranscript was seen in PBL, HPV, U937 and THP-1 cells; and about a 1.2kb transcript was seen in placenta, bone marrow and thyroid, and HPV andU937 cells. In all the tissues listed above, the signal intensity wasfaint. There appeared to be little expression in most normal tissues,suggesting that zcytor17 expression may be dependent on activation ofthe cell or tissues in which it is expressed.

B. Tissue Distribution in Tissue Panels Using PCR

A panel of cDNAs from human tissues was screened for zcytor17 expressionusing PCR. The panel was made in-house and contained 94 marathon cDNAand cDNA samples from various normal and cancerous human tissues andcell lines as shown below in Table 13. The cDNAs came from in-houselibraries or marathon cDNAs from in-house RNA preps, Clontech RNA, orInvitrogen RNA. The marathon cDNAs were made using the marathon-Ready™kit (Clontech, Palo Alto, Calif.) and QC tested with clathrin primersZC21195 (SEQ ID NO:78) and ZC21196 (SEQ ID NO:79) and then diluted basedon the intensity of the clathrin band. To assure quality of the panelsamples, three tests for quality control (QC) were run: (1) To assessthe RNA quality used for the libraries, the in-house cDNAs were testedfor average insert size by PCR with vector oligos that were specific forthe vector sequences for an individual cDNA library; (2) Standardizationof the concentration of the cDNA in panel samples was achieved usingstandard PCR methods to amplify full length alpha tubulin or G3PDH cDNAusing a 5′ vector oligo ZC14,063 (SEQ ID NO:48) and 3′ alpha tubulinspecific oligo primer ZC17,574 (SEQ ID NO:49) or 3′ G3PDH specific oligoprimer ZC17,600 (SEQ ID NO:50); and (3) a sample was sent to sequencingto check for possible ribosomal or mitochondrial DNA contamination. Thepanel was set up in a 96-well format that included a human genomic DNA(Clontech, Palo Alto, Calif.) positive control sample. Each wellcontained approximately 0.2-100 pg/μl of cDNA. The PCR reactions wereset up using oligos ZC26,358 (SEQ ID NO:80) and ZC26,359 (SEQ ID NO:81),TaKaRa Ex Taq™ (TAKARA Shuzo Co LTD, Biomedicals Group, Japan), andRediload dye (Research Genetics, Inc., Huntsville, Ala.). Theamplification was carried out as follows: 1 cycle at 94° C. for 2minutes, 35 cycles of 94° C. for 30 seconds, 66.3° C. for 30 seconds and72° C. for 30 seconds, followed by 1 cycle at 72° C. for 5 About 10 μlof the PCR reaction product was subjected to standard agarose gelelectrophoresis using a 4% agarose gel. The correct predicted DNAfragment size was observed in lymph node, prostate, thyroid, HPV(prostate epithelia), HPVS (prostate epithelia, selected), lung tumor,uterus tumor reactions, along with the genomic DNA reaction.

The DNA fragment for prostate tissue (2 samples), HPV (prostateepithelia), HPVS (prostate epithelia, selected), and genomic wereexcised and purified using a Gel Extraction Kit (Qiagen, Chatsworth,Calif.) according to manufacturer's instructions. Fragments wereconfirmed by sequencing to show that they were indeed zcytor17. TABLE 13Tissue/Cell line #samples Tissue/Cell line #samples Adrenal gland 1 Bonemarrow 3 Bladder 1 Fetal brain 3 Bone Marrow 1 Islet 2 Brain 1 Prostate3 Cervix 1 RPMI #1788 (ATCC 2 # CCL-156) Colon 1 Testis 4 Fetal brain 1Thyroid 2 Fetal heart 1 WI38 (ATCC # CCL-75 2 Fetal kidney 1 ARIP (ATCC1 # CRL-1674 - rat) Fetal liver 1 HaCat - human keratinocytes 1 Fetallung 1 HPV (ATCC # CRL-2221) 1 Fetal muscle 1 Adrenal gland 1 Fetal skin1 Prostate SM 2 Heart 2 CD3+ selected PBMC's 1 Ionomycin + PMAstimulated K562 (ATCC 1 HPVS (ATCC # CRL-2221) - 1 # CCL-243) selectedKidney 1 Heart 1 Liver 1 Pituitary 1 Lung 1 Placenta 2 Lymph node 1Salivary gland 1 Melanoma 1 HL60 (ATCC # CCL-240) 3 Pancreas 1 Platelet1 Pituitary 1 HBL-100 1 Placenta 1 Renal mesangial 1 Prostate 1 T-cell 1Rectum 1 Neutrophil 1 Salivary Gland 1 MPC 1 Skeletal muscle 1 Hut-102(ATCC # TIB-162) 1 Small intestine 1 Endothelial 1 Spinal cord 1 HepG2(ATCC # HB-8065) 1 Spleen 1 Fibroblast 1 Stomach 1 E. Histo 1 Testis 2Thymus 1 Thyroid 1 Trachea 1 Uterus 1 Esophagus tumor 1 Gastric tumor 1Kidney tumor 1 Liver tumor 1 Lung tumor 1 Ovarian tumor 1 Rectal tumor 1Uterus tumor 1C. Expression Analysis of Zcytor17 by PCR and Northern

Annotation of the cell types and growth conditions that affectexpression of the receptor is a useful means of elucidating its functionand predicting a source of ligand. To that end a wide variety of tissueand cell types were surveyed by PCR. The thermostable polymeraseAdvantage II™ (Clontech, La Jolla, Calif.) was used with theoligonucleotide primers ZC29,180 (SEQ ID NO:22) and ZC29,179 (SEQ IDNO:82) and 1-10 ng of the various cDNA templates listed below for 30amplification cycles of (94° C., 30 sec.; 66° C., 20 sec.; 68° C., 1min. 30 sec.). Following this, 20% of each reaction was run out on 0.8%agarose, TAE/ethidium bromide gels and visualized with UW light. Sampleswere then scored on the basis of band intensity. See Table 14 below.TABLE 14 Cells and Conditions Score 0-5 Hel stimulated with PMA 0 U937 3MCF-7 0 HuH7 1 Human follicle 0 HT-29 0 HEPG2 0 HepG2 stimulated withIL6 0 Human dermal endothelial 0 Human venous endothelial 0 Human CD4+ 0BEWO 0 Human CD19+ 1 Human PBMC stimulated with PHA, PMA, Ionomycin, 0IL2, IL4, TNFα 24 hours Human PBMC stimulated with LPS, PWM, 0 IFNγ,TNFα, 24 hours Human PBMC all of the above conditions for 48 hours 4HUVEC p.2 4 RPMI1788 0 TF1 0 Monkey spleen T cells stimulated with PMA,Ionomycin 0 Human prostate epithelia HPV transformed 5 Human tonsils,inflamed 0 HACAT 0 Human chondrocyte 1 Human synoviacyte 1 THP1 5 REH 0

Of the strong positive PCR signals, two were from the human monocytecell lines U937 and THP1.

These two cell lines along with a prostate epithelia line were selectedfor further analysis by Northern blot. Previous attempts at visualizinga transcript by northern analysis using mRNA from various tissuesyielded weak and diffuse signals in the surprisingly large size range of7-10 kb making this data difficult to interpret. A denaturingformaldehyde/MOPS/0.8% agarose gel was prepared (RNA Methodologies,Farrell, RE Academic Press) and 2 μg of polyA+ mRNA was run for eachsample along side an RNA ladder (Life Technologies, Bethesda, Md.). Thegel was then transferred to Hybond nylon (Amersham, Buckinghamshire,UK), UV crosslinked, and hybridized in ExpressHyb solution (Clontech,LaJolla, Calif.) at 68° C. overnight using a probe to human zcytoR17generated by PCR with the oligos ZC28,575 (SEQ ID NO:77), and ZC27,899(SEQ ID NO:19) and labeled with a Megaprime ³²P kit (Amersham). Thenorthern blot was subsequently washed with 0.2×SSC+01% SDS at 65 C for15 minutes and exposed to film for 7 days with intensifying screens. Aprominent 8 kb band was seen in both the prostate epithelia and U937lanes while a fainter band was present in the THP1 lane.

To optimize the cDNA used as a hybridization probe, four differentregions of the full-length human zcytoR17 sequence were amplified byPCR, labeled and hybridized as described above to southern blotscontaining genomic and amplified cDNA library DNA. The four probes,herein designated probes A-D, were amplified using the following primerpairs: (A) ZC28,575 (SEQ ID NO:77), ZC27,899 (SEQ ID NO:19); (B)ZC27,895 (SEQ ID NO:20), ZC28,917 (SEQ ID NO:83); (C) ZC28,916 (SEQ IDNO:84), ZC28,918 (SEQ ID NO:85); and (D) ZC28,916 (SEQ ID NO:84),ZC29,122 (SEQ ID NO:21). Human genomic DNA along with amplified cDNAlibraries demonstrated to contain zcytor17 by PCR were digested withEcoR1 and Xho1 to liberate inserts and run out on duplicate TAE/0.8%agarose gels, denatured with 0.5M NaOH, 1.5 M NaCl, blotted to Hybond,UV crosslinked and each hybridized with a distinct probe. Probe B wasfound to have the least nonspecific binding and strongest signal. Thus,Probe B was used for all subsequent hybridizations.

Given that the THP1 cells are an excellent model of circulatingmonocytes and expressed zcytor17 at low levels we treated them with avariety of compounds in an effort to increase expression of zcytoR17.The cells were grown to a density of 2e5/ml, washed and resuspended invarious stimulating media, grown for four or thirty hours, and harvestedfor RNA preparations. Each media was supplemented with one of thefollowing drugs or pairs of cytokines: LPS 2 ug/ml (Sigma Chemicals, St.Louis, Mo.), hTNFα 2 ng/ml (R&D Systems, Minneapolis, Minn.), hGM-CSF 2ng/ml (R&D Systems, Minneapolis, Minn.), hIFNγ 50 ng/ml (R&D Systems,Minneapolis, Minn.), hMCSF 1 ng/ml (R&D Systems, Minneapolis, Minn.),hIL6 1 ng/ml (R&D Systems, Minneapolis, Minn.), hIL1β 2 ng/ml (R&DSystems, Minneapolis, Minn.), hIFNγ 50 ng/ml+hIL4 0.5 ng/ml (R&DSystems, Minneapolis, Minn.), hIFNγ 50 ng/ml+hIL10 1 ng/ml (R&D Systems,Minneapolis, Minn.), PMA 10 ng/ml (Calbiochem, San Diego, Calif.) and anuntreated control. At the end of the culture period Total RNA wasprepared using an RNAeasy Midi-kit (Qiagen, Valencia, Calif.). Poly A+RNA was selected from the total RNA using an MPG kit (CPG, Lincoln Park,N.J.). Two micrograms of polyA+ RNA from each condition was run onformaldehyde/MOPS/agarose gels, transferred to nylon and UV crosslinkedas described above. These northern blots were then hybridized, as above,to probe B at 68° C. overnight, washed at high stringency with 0.2×SSC,0.1% SDS at 65 C, exposed to film overnight then exposed to phosphorscreens for signal quantitation. A dominant 8 kb mRNA as well arelatively weaker 2.8 kb band were seen in all lanes. A 20-fold increasein zcytor17 mRNA was seen in RNA from cells treated with hIFNγ for 30hours, this effect was slightly muted with simultaneous treatment withIL4. Minor 3 fold increases in mRNA were seen in RNA from cells treatedwith LPS, TNFα and GM-CSF while MCSF, IL6, and IL1β had no effect onzcytor17 mRNA levels. Taken together this data suggests a role for thezcytor17 receptor and its ligand in monocyte macrophage biology and byextension any number of disease processes in which these cellsparticipate.

Example 32 Tissue Distribution of Human Zcytor17 in Tissue Panels UsingNorthern Blot and PCR

A human zcytor17lig cDNA fragment was obtained using PCR with genespecific primers: Sense primer ZC41438 (SEQ ID NO:93) and antisenseprimer ZC41437 (SEQ ID NO:94) and template human zcytor17lig cDNA (SEQID NO:90) This fragment was purified using standard methods and about 25ng labeled with ³²P alpha dCTP using the Prime-It RmT random primerlabeling kit (Stratagene) and hybridized in Ultrahyb, (Ambion) and usedto expose Biomax film/intensifying screens per the manufacturer'srecommendations in each case. New, previously unused blots Including theClontech Human 12 lane MTN, the human brain MTN II, and the human brainMTN blot IV, the human immune system MTN II, and the human MTE array II,from Clontech were hybridized overnight at 42° C. per the Ambionultrahyb method. Non-specific radioactive counts were washed off using0.1 SSC/0.5% SDS at 55° C. The positive blots included the human 12 laneMTN (Clontech). Of the 12 tissues examined, only placenta was positivefor an approximately 1.2 KB transcript.

Example 33 Construction of Mammalian Expression Vectors that ExpressHuman Zcytor17lig-CEE

A. Construction of ZCytor17Lig-CEE/pZMP21

An expression plasmid containing all or part of a polynucleotideencoding zCytor17Lig-CEE (SEQ ID NO:95) was constructed via homologousrecombination. The plasmid was called zCytor17Lig-CEE/pZMP21.

The construction of zCytor17Lig-CEE/pZMP21 was accomplished bygenerating a zCytor17Lig-CEE fragment using PCR amplification. The DNAtemplate used for the production of the zCytor17Lig-CEE fragment waszCytor17Lig/pZP7nx. The primers used for the production of thezCytor17Lig-CEE fragment were: (1) ZC41,607 (SEQ ID NO:97) (sensesequence), which includes from the 5′ to the 3′ end: 28 bp of the vectorflanking sequence (5′ of the insert) and 21 bp corresponding to the 5′sequence of zCytor17Lig; and (2) ZC41,605 (SEQ ID NO:98) (anti-sensesequence), which includes from the 5′ to the 3′ end: 37 bp of the vectorflanking sequence (3′ of the insert), 3 bp of the stop codon, 21 bpencoding a C-terminal EE tag, and 21 bp corresponding to the 3′ end ofzCytor17Lig sequence. The fragment resulting from the above PCRamplification is a copy of the template zCytor17Lig with the addition ofa C-terminal EE tag, yielding a final product zCytor17Lig-CEE.

PCR reactions were run as follows: To a 100 μl final volume was added:10 μl of 10× Taq Polymerase Reaction Buffer with 15 mM MgCl (Gibco), 1μl of Taq DNA Polymerase (5 units/μl, Gibco), 3 μl of 10 mM dNTPs, 78 μldH₂O, 3 μl of a 20 pmol/μl stock of primer ZC41,607 (SEQ ID NO:97) 3 μlof a 20 pmol/μl stock of primer ZC41,605 (SEQ ID NO:98), and 2 μl of a0.13 μg/μl stock of zCytor17lig template DNA. A volume equal to 50 μl ofmineral oil was added to the mixture. The reaction was heated to 94° C.for 5 minutes, followed by 35 cycles at 94° C. for 1 minute; 55° C. for2 minutes; 72° C. for 3 minutes; followed by a 10 minute extension at72° C. and held at 4° C. until the reaction was collected.

The plasmid pZMP21 was restriction digested with BglII enzyme, cleanedwith a QiaQuick PCR Purification Kit (Qiagen) using a microcentrifugeprotocol, and used for recombination with the PCR fragment. PlasmidpZMP21 was constructed from pZMP20 which was constructed from pZP9(deposited at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, and is designated No. 98668) withthe yeast genetic elements from pRS316 (deposited at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, and designated No. 77145), an IRES element from poliovirus,and the extracellular domain of CD8, truncated at the carboxyl terminalend of the transmembrane domain. PZMP21 is a mammalian expression vectorcontaining an expression cassette having the MPSV promoter,immunoglobulin signal peptide intron, multiple restriction sites forinsertion of coding sequences, a stop codon and a human growth hormoneterminator. The plasmid also has an E. coli origin of replication, amammalian selectable marker expression unit having an SV40 promoter,enhancer and origin of replication, a DHFR gene, the SV40 terminator, aswell as the URA3 and CEN-ARS sequences required for selection andreplication in S. cerevisiae.

Fifty microliters of competent yeast cells (S. cerevisiae) wereindependently combined with 100 ng of cut plasmid, 5 μl of previouslydescribed PCR mixture, and transferred to a 0.2 cm electroporationcuvette. The yeast/DNA mixture was electropulsed at 0.75 kV (5 kV/cm), ∞ohms, 25 μF. Each cuvette had 600 μl of 1.2 M sorbitol added, and theyeast was plated in one 100 μl aliquot and one 300 μl aliquot onto twoURA-D plates and incubated at 30° C. After about 72 hours, the Ura+yeast transformants from a single plate were resuspended in 1 ml H₂O andspun briefly to pellet the yeast cells. The cell pellet was resuspendedin 500 μl of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mMTris, pH 8.0, 1 mM EDTA). The 500 μl of the lysis mixture was added toan Eppendorf tube containing 300 μl acid washed 600 μm glass beads and300 μl phenol-chloroform, vortexed for 1 minute intervals two or threetimes, followed by a 5 minute spin in a Eppendorf centrifuge at maximumspeed. Three hundred microliters of the aqueous phase was transferred toa fresh tube, and the DNA precipitated with 600 μl 100% ethanol (EtOH),followed by centrifugation for 10 minutes at 4° C. The DNA pellet wasthen washed with 500 μl 70% EtOH, followed by centrifugation for 1minute at 4° C. The DNA pellet was resuspended in 30 μl H₂O.

Transformation of electrocompetent E. coli cells (MC1061) was done with5 μl of the yeast DNA prep and 50 μl of MC1061 cells. The cells wereelectropulsed at 2.0 kV, 25 μF and 400 ohms(Ω). Followingelectroporation, 600 μl SOC (2% Bacto' Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO₄, 20 mM glucose) was added. The electroporated E. coli cells wereplated in a 200 μl and a 50 μl aliquot on two LB AMP plates (LB broth(Lennox), 1.8% Bacto Agar (Difco), 100 mg/L Ampicillin). The plates wereincubated upside down for about 24 hours at 37° C. ThreeAmpicillin-resistant colonies were selected at random and submitted forsequence analysis of the insert. Large-scale plasmid DNA was isolatedfrom a sequence-confirmed clone using the Qiagen Maxi kit (Qiagen)according to manufacturer's instructions.

B. Construction of Mouse ZCytor17Lig(m)-CEE/pZMP21

An expression plasmid containing the entire polynucleotide encodingmurine zCytor17Lig-CEE (SEQ ID NO:104 and SEQ ID NO:105) was alsoconstructed via homologous recombination using the method described inExample 33A above. The primers used were: (1) ZC41643 (SEQ ID NO:106)(forward, 5′ to 3′ sense) having a 28 bp vector overlap 5′ of theinsertion point; 21 bp of the 5′ end of zcytor17lig(m) and (2) ZC41641(SEQ ID NO:107) (reverse, 5′ to 3′ antisense) having a 37 bp vectoroverlap 3′ of the insertion point; 3 bp stop codon; 21 bp C-terminal EEtag; 24 bp of the 3′ end of zCytor17Lig(m)-CEE. The plasmid was calledzcytor17lig(m)-CEE/pZMP21. The polynucleotide sequence ofzcytor17lig(m)-CEE is shown in SEQ ID NO:104, and correspondingpolypeptide sequence is shown in SEQ ID NO:105.

Example 34 Transfection and Expression of Zcytor17lig-CEE Polypeptides

A. Expression of Human ZCytor17Lig-CEE/pZMP21 in 293T Cells

ZCytor17Lig-CEE was expressed transiently in 293T cells (StanfordUniversity School of Medicine, Stanford, Calif.; ATCC No. SD-3515) togenerate initial purified protein. The day before the transfection, 293Tcells were seeded at 6.5×10⁴ cells/cm² in 30 T162 culture flasks with atotal volume of 30 ml of culture media (SL7V4+5% FBSμ+1% Pen/Strep) perflask. The cells were allowed to incubate for 24 hours at 37° C.

A DNA/Liposome mixture was prepared as follows: Two 50 ml conical tubeswere filled with 25 mLs of transfection media (SL7V4+1% Pen/Strep) and1.13 mg of zCytor17Lig-CEE/pZMP21 (Example 33) was added to each. Aseparate set of two 50 ml conical tubes were filled with 22 ml oftransfection media (above) and 3 ml of liposomes (Lipofectamine, Gibco)was added to each. For each set of tubes, one tube of DNA was added toone tube of liposomes and the DNA/liposome mix was incubated for 30minutes. The two 50 ml conical tubes containing the DNA/liposomemixtures were pooled (about 100 ml) and 300 ml of transfection media wasadded.

The 30 flasks of the 293T cells were decanted, washed 1× with about 15ml of PBS, and 12.5 ml of the diluted DNA/liposome mixture was added toeach flask. The flasks were incubated for 3 hours at 37° C. After theincubation period, 25 ml of culture media (above) were added to eachT162 flask. The transfection media was harvested after approximately 96hours and was used for protein purification (Example 35).

B. Expression of Mouse ZCytor17Lig-CEE(m)/pZMP21 in 293T Cells

Mouse zCytor17Lig(m)-CEE was expressed transiently in 293T cells asdescribed in Example 34A and cultured media was used for proteinpurification (Example 35).

Example 35 Purification of Zcytor17lig-CEE from 293T Cells

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying both mouse and humanZcytor17lig containing C-terminal Glu-Glu (EE) tags (SEQ ID NO:103).Conditioned media from 293T cells expressing Zcytor17lig-CEE (Example34) was purified. Total target protein concentrations of the conditionedmedia were determined via SDS-PAGE and Western blot analysis with theanti-EE antibody.

A 5.5 ml column of anti-EE Poros 50 A (PE BioSystems, Framingham, Mass.)(prepared as described below) was poured in a Waters AP-1, 1 cm×7 cmglass column (Waters, Milford, Mass.). The column was flow packed andequilibrated on a BioCad Sprint (PE BioSystems, Framingham, Mass.) withphosphate buffered saline (PBS) pH 7.4. The conditioned media wasadjusted with NaCl to 0.3 M and the pH adjusted to 7.2. The conditionedmedia was then loaded on the column overnight with about 3 ml/minuteflow rate. The column was washed with 10 column volumes (CVs) of PBS pH7.4, and again washed with 3 CVs 5× Sigma PBS pH 7.4. It was step elutedwith 0.5 M Acetate, 0.5 M NaCl, pH 2.5 at 3 ml/minute. The fractiontubes contained 1 ml Tris base (no pH adjustment) to neutralize theelution immediately. The column was again washed for 2 CVs with 5× SigmaPBS, pH 7.4 to neutralize the column and then equilibrated in PBS (pH7.4). Two ml fractions were collected over the entire elutionchromatography and absorbance at 280 and 215 nM were monitored; the passthrough and wash pools were also saved and analyzed. The 5× PBS and theacid elution peak fractions were analyzed for the target protein viaSDS-PAGE Silver staining and Western Blotting with the primary antibodyanti-EE and secondary antibody, anti mouse-HRP conjugated. The acidelution fractions of interest were pooled and concentrated from 38 ml to0.8 ml using a 5000 Dalton molecular weight cutoff membrane spinconcentrator (Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

To separate Zcytor17lig-CEE from aggregated material and any othercontaminating co-purifying proteins, the pooled concentrated fractionswere subjected to size exclusion chromatography on a 1.6×60 cm (120 ml)Superdex 75 (Pharmacia, Piscataway, N.J.) column equilibrated and loadedin PBS at a flow rate of 1.0 ml/min using a BioCad Sprint. Threemilliliter fractions were collected across the entire chromatography andthe absorbance at 280 and 215 nM were monitored. The peak fractions werecharacterized via SDS-PAGE Silver staining, and only the most purefractions were pooled. This material represented purified Zcytor17lig-CEE protein.

On Western blotted, Coomassie Blue and Silver stained SDS-PAGE gels, theZcytor17lig-CEE was one major band. The protein concentration of thepurified material was performed by BCA analysis (Pierce, Rockford, Ill.)and the protein was aliquoted, and stored at −80° C. according tostandard procedures.

To prepare PorosA50 anti-EE, a 65 ml bed volume of Poros A50 (PEBiosystems) was washed with 100 ml of water and then 0.1 Mtriethanolamine, pH 8.2 (TEA, ICN, Aurora, Ohio), 1 M Na₂S0₄, pH 8.8containing 0.02% sodium azide using a vacuum flask filter unit. The EEmonoclonal antibody solution, at a concentration of 2 mg/ml in a volumeof 300 ml, was mixed with the washed resin in a volume of 250 ml. Afteran overnight incubation at room temperature, the unbound antibody wasremoved by washing the resin with 5 volumes of 200 mM TEA, 1 M Na₂S0₄,pH 8.8 containing 0.02% sodium azide as described above. The resin wasresuspended in 2 volumes of TEA, 1 M Na₂S0₄, pH 8.8 containing 0.02%sodium azide and transferred to a suitable container. Three ml of 25mg/ml (68 mM) Disuccinimidyl suberate (in DMSO supplied by Pierce,Rockford, Ill.) is added and the solution is incubated for three hoursat room temperature. Nonspecific sites on the resin were then blocked byincubating for 10 min at room temperature with 5 volumes of 20 mMethanolamine (Sigma, St. Louis, Mo.) in 200 mM TEA, pH 8.8 using thevacuum flask filter unit. The resin is washed with PBS, pH 7.4, followedby 0.1 M Glycine, pH 3 and then neutralized with 10× PBS. After washingwith distilled water, the final coupled anti-EE Poros-A 50 resin wasstored at 4° C. in 20% Ethanol.

Example 36 N-terminal Sequencing of Human and Mouse Zcytor17lig

A. N-Terminal Sequencing of Human Zcytor17lig

Standard automated N-terminal polypeptide sequencing (Edman degradation)was performed using reagents from Applied Biosystems. N-terminalsequence analysis was performed on a Model 494 Protein Sequencer System(Applied Biosystems, Inc., Foster City, Calif.). Data analysis wasperformed with Model 610A Data Analysis System for Protein Sequencing,version 2.1a (Applied Biosystems).

A purified human zcytor17lig-CEE sample (Example 35) was supplied. Thesample was loaded onto a prepared glass fiber filter for n-terminalsequencing. The glass fiber filter was prepared by precycling it withBiobrene™.

N-terminal sequence analysis of the secreted human zcytor17ligpolypeptide did not verify the predicted cleavage site of the signalsequence but resulted in a mature start at residue 27(Leu) in SEQ IDNO:2 of the human zcytor17lig precursor sequence.

B. N-Terminal Sequencing of Human Zcytor17lig

Standard automated N-terminal polypeptide sequencing (Edman degradation)was performed using reagents from Applied Biosystems. N-terminalsequence analysis was performed on a Model 494 Protein Sequencer System(Applied Biosystems, Inc., Foster City, Calif.). Data analysis wasperformed with Model 610A Data Analysis System for Protein Sequencing,version 2.1a (Applied Biosystems).

A purified mouse zcytor17lig-CEE sample was supplied as captured onProtein G Sepharose/anti-EE beads (Example 35). The beads were placed inreducing SDS PAGE sample buffer and on a boiling water bath beforerunning on SDS PAGE, using a Novex SDS PAGE system (4-12% Bis-Tris MESNuPAGE; Invitrogen) as per manufacturer's instructions. The gel waselectrotransferred to a Novex PVDF membrane (Invitrogen), and Coomassieblue stained (Sigma, St. Louis, Mo.) using standard methods.Corresponding anti-EE Western blots were performed to identify thezcytor17lig band for N-terminal protein sequencing. The mouse anti-EEIgG HRP conjugated antibody used was produced in house.

N-terminal sequence analysis of the secreted mouse zcytor17ligpolypeptide verified the predicted cleavage site of the signal sequenceresulting in a mature start at 31 (Ala) in reference to SEQ ID NO:11 andSEQ ID NO:91 of the mouse zcytor17lig precursor sequence.

Example 37 Cos Cell Binding Assay

A binding assay was used to test the binding of the zcytor17lig toreceptors comprising zcytor17 receptor, such as the zcytor17 receptor orreceptor heterodimers and trimers comprising zcytor17 receptor (e.g.,zcytor17/OSMR, zcytor17/WSX-1, or zcytor17/OSMR/WSX-1, or other Class Icytokine receptor subunits). Zcytor17 receptor plasmid DNA wastransfected into COS cells and transfected COS cells were used to assessbinding of the zcytor17lig to receptors comprising zcytor17 receptor asdescribed below.

A. COS Cell Transfections

The COS cell transfection was performed as follows: Mix 800 ng receptorplasmid DNA in the following combinations: pZp7pX/zcytor17 alone;pZp7Z/WSX-1 alone; pZp7NX/OSMR alone; pZp7pX/zcytor17+pZp7NX/OSMR;pZp7pX/zcytor17+pZp7Z/WSX-1; pZp7NX/OSMR+pZp7Z/WSX-1;pZp7pX/zcytor17+pZp7NX/OSMR+pZp7Z/WSX-1) and 4 ul Lipofectamine™ in 80μl serum free DMEM media (55 mg sodium pyruvate, 146 mg L-glutamine, 5mg transferrin, 2.5 mg insulin, 1 μg selenium and 5 mg fetuin in 500 mlDMEM), incubate at room temperature for 30 minutes and then add 320 μlserum free DMEM media. Add this 400 ul mixture onto 2 ×10⁵ COScells/well plated on 12-well tissue culture plate (fibronectin-coated)and incubate for 5 hours at 37° C. Add 500 ul 20% FBS DMEM media (100 mlFBS, 55 mg sodium pyruvate and 146 mg L-glutamine in 500 ml DMEM) andincubate overnight.

B. Binding Assay

The binding assay was performed as follows: media was rinsed off cellswith PBS+0.1% BSA, and then cells were blocked for 60 minutes with thesame solution. The cells were then incubated for 1 hour in PBS+0.1% BSAwith 1.0 μg/ml zcytor17ligCEE purified protein. Cells were then washedwith PBS+0.1% BSA and incubated for another hour with 1:1000 dilutedmouse anti-GluGlu antibody. Again cells were washed with PBS+0.1% BSA,then incubated for 1 hour with 1:200 diluted goat anti-mouse-HRPconjugated antibody.

Positive binding was detected with fluorescein tyramide reagent diluted1:50 in dilution buffer (EN kit) and incubated for 4-6 minutes, andwashed with PBS+0.1% BSA. Cells were fixed for 15 minutes with 1.8%Formaldehyde in PBS, then washed with PBS+0.1%BSA. Cells were preservedwith Vectashield Mounting Media (Vector Labs Burlingame, Calif.) diluted1:5 in PBS. Cells were visualized using a FITC filter on fluorescentmicroscope.

Positive binding was detected for cells transfected with zcytor17 only,zcytor17+OSMRbeta, zcytor17+WSX-1, and zcytor17+OSMRbeta+WSX-1. Nobinding was detected for cells transfected with WSX-1+OSMRbeta, withOSMRbeta only, or with WSX-1 only.

Example 38 Mouse Zcytor17lig Activates Mouse Zcytor17/OSMRbetaReceptor-In Luciferase Assay

A. Cloning of Full-Length Mouse Zcytor17 and Mouse OSMRbeta forExpression

A mouse testes cDNA library was screened for a full-length clone ofmouse zcytoR17. The library was plated at 65,500 cfu/plate on 24 LB+Ampplates. Filter lifts were prepared using Hybond N (Amersham-PharmaciaBiotech, Inc., Piscataway, N.J.) on a total of approximately 1.6 millioncolonies. The filters were marked with a hot needle for orientation andthen denatured for 6 minutes in 0.5 M NaOH and 1.5 M Tris-HCl, pH 7.2.The filters were then neutralized in 1.5 M NaCl and 0.5 M Tris-HCl, pH7.2 for 6 minutes. The DNA was affixed to the filters using a UVcrosslinker (Stratalinker®, Stratagene, La Jolla, Calif.) at 1200joules. The filters were then left to dry overnight at room temperature.

The next day, the filters were pre-washed at 65° C. in pre-wash bufferconsisting of 0.25×SSC, 0.25% SDS and 1 mM EDTA. Cell debris wasmanually removed using Kimwipes® (Kimberly-Clark) and the solution waschanged 3 times over a period of 1 hour. Filters were air dried andstored at room temperature until needed. The filters were thenprehybridized for approximately 3 hours at 63° C. in 20 ml ofExpressHyb™Hybridization Solution (Clontech, Palo Alto, Calif.).

Probe B (Example 31) was generated by PCR from human zcytoR17 templateusing oligonucleotide primers ZC27,895 (SEQ ID NO:20) and ZC28,917 (SEQID NO:83) and was radioactively labeled with ³²P using a commerciallyavailable kit (Megaprime DNA Labeling System; Amersham PharmaciaBiotech, Piscataway, N.J.) according to the manufacturer's instructions.The probe was purified using a Stratagene™ push column (NucTrap® column;Stratagene, La Jolla, Calif.). The probe was denatured at 100° C. for 15min and added to ExpressHyb™. Filters were hybridized in 15 mlhybridizing solution containing 1.6×10⁶ cpm/ml of probe at 63° C.overnight. Filters were washed at 55° C. in 2×SSC, 0.1% SDS and 1 mMEDTA and exposed to X-ray film at −80° C. for 4 1/2 days. Thirteenpositives were picked from the plates as plugs and placed in 1 ml LB+amp in 1.7 ml tubes. Tubes were placed at 4° C. overnight. These 13positives were subjected to two further rounds of purification. Thetertiary plates were outgrown at 37° C. after filter lifts were takenand single colonies were picked and sent to sequencing. Three of thesewere determined to contain sequence of the mouse ortholog of zcytoR17.

In addition, a PCR product was generated using CTLL-2 cDNA as a templateand oligonucleotides ZC38,239 (SEQ ID NO:123) and ZC38,245 (SEQ IDNO:124) as primers. CTLL-2 is a mouse cytotoxic T lymphocyte cell line(ATCC No. TIB-214). This PCR reaction was run as follows: 1 cycle at 95°C. for 1 minute, 30 cycles at 95° C. for 15 seconds, 68° C. for 3minutes, then 68° C. for 10 minutes; 4° C. soak. The PCR reaction usedapproximately 0.5 ng of cDNA, 20 pmoles of each oligonucleotide, and 1μl of Advantage II polymerase mix (ClonTech). About 6% of the PCRproduct was used as a template in a new PCR reaction, as above, exceptwith oligonucleotides ZC38,239 (SEQ ID NO:123) and ZC38,238 (SEQ IDNO:125). This PCR reaction was run as follows: 30 cycles at 94° C. for45 seconds, 65° C. for 45 seconds, 72° C. for 1 minute then 72° C. for 7minutes; 10° C. soak. Most of the PCR reaction was loaded on a 1.0%agarose gel and the predominant band at approximately 360 bp wasexcised, the DNA fragment was eluted, and DNA sequencing was performed.

The sequence of the mouse zcytor17 polynucleotide is shown in SEQ IDNO:126 and the corresponding amino acid sequence shown in SEQ ID NO:127.In addition, a truncated soluble form of the mouse zcytor17polynucleotide is shown in SEQ ID NO:128 and the corresponding aminoacid sequence shown in SEQ ID NO:129.

To obtain a full-length mouse OSMRbeta cDNA, 5′ and 3′ PCR products wereisolated and joined using an internal BamHI site. The PCR primers weredesigned using the nucleotide sequence SEQ ID NO:134 and include EcoRIand XbaI restriction sites for cloning purposes. The genomic mouseOSMRbeta nucleic acid sequence is shown in SEQ ID NO:134, wherein thecoding sequence encompasses residues 780 to 3692 encoding a mouseOSMRbeta 970 amino acid polypeptide, which is shown in SEQ ID NO:135. Adegenerate nucleic acid sequence which encodes the polypeptide of SEQ IDNO:135 is shown in SEQ ID NO:136.

A 5′ PCR product was generated using an in-house 3T3-L1 (differentiatedmouse adipocyte) cDNA library as a template and oligonucleotidesZC41,764 (SEQ ID NO:130) and ZC41,598 (SEQ ID NO:131) as primers. This5′ PCR reaction was run as follows: 30 cycles at 95° C. for 45 seconds,55° C. for 45 seconds, 72° C. for 1 minute 30 seconds, then 72° C. for 7minutes; 4° C. soak. The PCR reaction used approximately 3 μg of plasmidprepared from the cDNA library, 20 pmoles of each oligonucleotide, andfive units of Pwo DNA polymerase (Roche). About 90% of the 5′ PCRproduct was digested with EcoRI and BamHI and gel purified on a 1.0%agarose gel. The approximately 1446 bp band was excised and used forligation (see below).

A 3′ PCR product was generated using a mouse placenta in-house cDNAlibrary as a template and oligonucleotides ZC41,948 (SEQ ID NO:132) andZC41,766 (SEQ ID NO:133) as primers. This 3′ PCR reaction was run asfollows: 30 cycles at 95° C. for 45 seconds, 55° C. for 45 seconds, 72°C. for 1 minute 30 seconds, then 72° C. for 7 minutes; 4° C. soak. ThePCR reaction used approximately 3 μg of plasmid prepared from the cDNAlibrary, 20 pmoles of each oligonucleotide, and five units of Pwo DNApolymerase (Roche). About 90% of the 3′ PCR product was digested withBamHI and XbaI and gel purified on a 1.0% agarose gel. The approximately2200 bp band was excised and used for ligation along with the 5′ PCRproduct (described above) to the expression vector pZP-5Z digested withEcoRI and XbaI. The three-part ligation was performed with the 5′ EcoRIto BamHI fragment above, the 3′ BamHI to XbaI fragment, and theexpression vector pZP-5Z digested with EcoRI and XbaI. This generated apZP-5Z plasmid containing a full-length cDNA for mouse OSMRbeta(nucleotides 780 to 3692 of SEQ ID NO:134), designated pZP-5Z/OSMRbeta.The full length mouse OSMRbeta cDNA in pZP5Z/OSMRbeta has two amino acidinsertions from SEQ ID NO:135. There is a duplication of amino acidGlycine at position 370 and a duplication of amino acid Glutamic Acid atposition 526. Plasmid pZP-5Z is a mammalian expression vector containingan expression cassette having the CMV promoter, multiple restrictionsites for insertion of coding sequences, and a human growth hormoneterminator. The plasmid also has an E. coli origin of replication, amammalian selectable marker expression unit having an SV40 promoter,enhancer and origin of replication, a zeocin resistance gene and theSV40 terminator.

The resulting transformants were sequenced to confirm the mouse OSMRbetacDNA sequence.

B. Construction of BaF3/KZ134/Zcytor17m, BaF3/KZ134/Zcytor17m/OSMRbetam,BHK/KZ134/Zcytor17m, and BHK/KZ134/Zcytor17m/OSMRbetam Cell Lines

Stable BaF3/KZ134 and BHK/KZ134 cell lines (Example 20) were transfectedwith an expression plasmid encoding full-length mouse zcytor17,pZP-7P/zcytor17m (Example 38A), to create BaF3/KZ134/zcytor17m andBHK/KZ134/zcytor17m cells, respectively. The mouse OSMRbeta expressionplasmid, pZP-5Z/OSMRbetam (Example 38A), was then transfected into thesecells to create BaF3/KZ134/zcytor17m/OSMRbetam andBHK/KZ134/zcytor17m/OSMRbetam cell lines, respectively. Methods were asdescribed in Example 4 with the exception that Baf3/KZ134/zcytor17m andBHK/KZ134/zcytor17m were selected with, in addition to Geneticin, 2ug/ml puromycin while Baf3/KZ134/zcytor17m/OSMRbetam andBHK/KZ134/zcytor17m/OSMRbetam were selected with, in addition toGeneticin, 2 ug/ml puromycin and 200 ug/ml zeocin.

Clones were diluted, plated and selected using standard techniques.Clones were screened by luciferase assay (see Example 20, above) usingthe mouse zcytor17lig conditioned media or purified mouse zcytor17ligprotein (Example 35) as an inducer. Clones with the highest luciferaseresponse (via STAT luciferase) and the lowest background were selected.Stable transfectant cell lines were selected.

C. Mouse Zcytor17lig Activates Mouse Zcytor17 Receptor inBaF3/KZ134/Zcytor17m/OSMRbetam or BHK/KZ134/Zcytor17m/OSMRbetamLuciferase Assay

Cell lines were plated for luciferase assays as described in Example 20above. STAT activation of the BaF3/KZ134/Zcytor17m,BaF3/KZ134/zcytor17m/OSMRbetam, BHK/KZ134/zcytor17m, orBHK/KZ134/zcytor17m/OSMRbetam cells was assessed using (1) conditionedmedia from BHK570 cells transfected with the human zcytor17lig (Example7), (2) conditioned media from BHK570 cells transfected with the mousezcytor17lig (Example 18), (3) purified mouse and human zcytor17lig(Example 35), and (4) mIL-3 free media to measure media-only controlresponse. Luciferase assays were performed as described in Example 20.

The results of this assay confirm the STAT reporter response of theBaF3/KZ134/zcytor17m/OSMRbetam and BHK/KZ134/zcytor17m/OSMRbetam cellsto the mouse zcytor17lig when compared to either theBaF3/KZ134/zcytor17m cells, the BHK/KZ134/zcytor17m cells or theuntransfected BaF3/KZ134 or BHK/KZ134 control cells, and show that theresponse is mediated through the mouse zcytor17/OSMRbeta receptors. Theresults also show that the human zcytor17lig does not activate the STATreporter assay through the mouse receptor complex.

Example 39 Human Zcytor17 Ligand Binding to Zcytor17 andZcytor17/OSMRbeta by Flow Cytometry

The biotinylation of human zcytor17 L was done as follows: 100 μL ofzcytor17 at 5.26 mg/mL was combined with 30μL of 10 mg/mL EZ-linkSulfo-NHS-LC-biotin (Pierce, Rockford, Ill.) dissolved in ddH₂O. Thissolution was incubated on a rocker for 30 minutes at room temperature.After biotinylation the solution was dialyzed in PBS using aSlide-A-Lyzer dialysis cassette.

To test the binding properties of human zcytor17 ligand to differentreceptor combinations both BHK and BAF3 cells were transfected withexpression plasmids using standard techniques well-known in the art.These plasmids were transfected into both cell lines in the followingcombinations: zcytor17 alone, OSMRbeta alone, and both zcytor17 andOSMRbeta. Transfection was performed as detailed above. UntransfectedBHK and BAF3 cells were used as controls. Cells were stained by FACS asfollows: 2E5 cells were stained with either: 2.0 μg/mL, 100 ng/mL, 10ng/mL, 1.0 ng/mL, 100 pg/mL, 10 pg/mL, 1.0 pg/mL of biotinylatedzcytor17 L or left unstained for 30 minutes on ice in FACS buffer(PBS+2% BSA+2% NHS (Gemini)+2% NGS). Cells were washed 1.5 times andthen stained with SA-PE (Jackson Immuno Laboratories) at 1:250 for 30minutes on ice. Cells were then washed 1.5 times with FACS buffer andresuspended in FACS buffer and analyzed by FACS on a BD FACSCaliberusing CellQuest software (Becton Dickinson, Mountain View, Calif.).

Both BHK and BAF3 cells showed that zcytor17 ligand bound to bothzcytor17 alone and in combination with OSMRbeta with the binding to thezcytor17/OSMRbeta heterodimer being slightly stronger. No binding wasseen in either cell lines expressing OSMRbeta alone. The zcytor17 ligandbound in a concentration dependent manner. The mean fluorescentintensity (MFI) values for the BHK binding are shown below in Table 15.TABLE 15 zcytor17 μg/mL 2.0 0.100 0.010 0.001 0.0001 0.00001 0.0000010.0 BHK C17 + OSMRbeta 3780 2126 328 53 17 15 14 13 BHK-C17 3032 1600244 39 16 15 14 15 BHK-OSMRbeta 13 X X X X X X  0 BHK-WT 15  14  13 X XX X 13 zcytor17 μg/mL 10.0 3.33 1.11 0.37 0.12 0.04 0.00 BAF3-C17 +OSMRbeta 531 508 489 441 364 247 7 BAF3-OSMRbeta 6 5 5 5 5 5 11 BAF3-WT13 13 12 12 12 12 13 zcytor17 ng/mL 100.0 10.0 1.0 0.0 BAF3-C17 347 7217 7

Example 40 Gene Expression Array Analysis of Human Zcytor17lig TreatedCells

RNA was isolated from human zcytor17lig treated A549 cells,

zcytor17lig treated SK-LU-1 cells, and untreated control cells using aRNeasy Midi Kit (Qiagen, Valencia, Calif.) according to the manufacturesinstructions.

Gene expression profiling of the cells treated with zcytor17lig and the

respective control cells was carried out using GEArray Q series cDNAexpression arrays (SuperArray Inc., Bethesda, Md.). The Q Series cDNAexpression arrays contain up to 96 cDNA fragments associated with aspecific biological pathway, or genes with similar functions orstructural features. Comparison of arrays from treated and control cellsallows for a determination of the up and down regulation of specificgenes. Probe labeling, hybridization and detection were carried outaccording to the manufactures instructions. Chemiluminscent signaldetection and data acquisition was carried out on a Lumi-Imagerworkstation (Roche, Indianapolis, Ind.). The resulting image data wasanalyzed using ImageQuant 5.2 (Amersham Biosciences, Inc., Piscataway,N.J.) and GEArray Analyzer 1.2 (SuperArray Inc., Bethesda, Md.)software.

Analysis of the results from the Human Interleukin and Receptor Q

series HS-014N arrays, showed, after normalization, an approximate 4.7fold increase of IL13RA2 signal in the zcytor17lig treated human SK-LU-1cells and an approximate 2.2 fold increase of the IL13RA2 signal in thezcytor17lig treated human A549 cells.

These results indicate that zcytor17lig significantly up regulated

IL13RA2 in the SK-LU-1 and A549 cells. Both of these are establishedcell lines derived from human lung carcinomas (Blobel et al., VirchowsArch B Cell Pathol Incl Mol Pathol., 1984;45(4):407-29). Morespecifically, A549 is characterized as a human pulmonary epithelial cellline (Lin, et al., J Pharm Pharmacol., September 2002;54(9):1271-8;Martinez et al., Toxicol Sci., October 2002;69(2):409-23).

Interleukin-13 (IL13), a cytokine secreted by activated T lymphocytes,has been demonstrated to be both necessary and sufficient for theexpression of allergic asthma and for use in experimental models ofasthma, which include airway hyperresponsiveness, eosinophilrecruitment, and mucus overproduction (Wills-Karp et al., Science,1998;282:2258-2261). It has been shown, that selective neutralization ofIL13 will ameliorate the asthma phenotype (Grunig et al., Science, 1998;282:2261-2263). It has also been reported that IL13 is involved in theup regulation of mucin gene MUC8 expression in human nasal polypepithelium and cultured nasal epithelium (Kimm et al., ActaOtolaryngol., September 2002;122(6):638-643; Seong et al., Acta _(—)Otolaryngol., June 2002;122(401-407). MUC8, a major airway mucinglycoprotein, is implicated as playing a role in the pathogenesis ofmucus hypersecretion in chronic sinusitis with polps (Seong et al., ActaOtolaryngol., June 2002;122(4):401 -407).

Functionally, IL13 signals through a receptor complex consisting of theinterleukin-13 receptor alpha-1 chain (IL13RA1) and IL-4 receptor alpha(IL4RA) (Daines and Hershey, J Biol Chem., 2002; 22(12):10387-10393). Ithas also been shown, that the interleukin-13 receptor alpha-2 (IL13RA2)binds IL13 with high affinity, but by itself (Daines and Hershey, J BiolChem., 2002; 22(12):10387-10393). This receptor lacks, however, thecytoplasmic domain necessary for signaling and, therefore, is consideredto be a decoy receptor. It has been shown that IL13RA2 is predominatelyan intracellular molecule that can be quickly mobilized fromintracellular stores and surface expressed following cellular treatmentwith interferon (IFN)-gamma. The surface expression of IL13RA2 afterIFN-gamma treatment does not involve protein synthesis and results indiminished IL13 signaling (Daines and Hershey, J Biol Chem., 2002;22(12):10387-10393).

The results of the gene expression array analysis for zcytor17ligindicate the action of zcytor17lig to be novel to that of IFN-gamma inthat the zcytor17lig treatment of lung epithelial derived cell linesresulted in a significant increase of IL13RA2 gene expression. It isconceivable, therefore, that zcytor17lig treatment would be beneficialin cases where long-term up regulation of IL13RA2 expression and downregulation of IL13 is desired such as in asthma, airway hyperactivity(AHR), and mucin regulation, including chronic sinusitis with polps.

Example 41 Murine Zcytor17lig Trangenic Mice

To evaluate the in vivo effects of zcytor17lig overexpression, multiplefounders of transgenic mice expressing the murine form of the gene weregenerated, driven by two different promoters: the lymphocyte-specificpromoter Eμ/lck, and the ubiquitous promoter, EF1α (Example 22). Serumprotein levels range from approximately 20-300 ng/ml. The Eμ/lckpromoter generated mice with higher levels of serum protein than thosein the EF1α-zcytor17lig transgenic mice.

The zcytor17lig transgenic mice developed a skin phenotype around 4-8weeks of age. The fur of the transgenic mice became “ruffled,” withobvious piloerection and mild to severe hair loss, usually on theirbacks, sides of the torso, and around their eyes. This phenotype wasconsistently found in mice with detectable levels of zcytor17lig proteinin their serum. Among the founders, 100% incidence rate among the miceexpressing the Eμ/lck-driven gene, and a 50% incidence in theEF1α-zcytor17lig transgenic mice was noted, correlating well with therelative levels of zcytor17lig that was detected in their serum. Thetransgenic skin appeared to be pruritic, as evidenced by the scratchingbehavior of the mice, sometimes excessive enough to induce excoriationand lesions of the skin, which usually became infected (with at leastStaphylococcus aureus). The mice were originally identified with metalear tags, but in most cases, the ear tags were forcibly removed by themice themselves. This often resulted in severe damage to the externalear. These wounded ears often did not heal properly, as reflected in thepresence of long-lasting pustules and crusting, and a seeping, expandingwound would that developed in many of the animals, behind and betweentheir ears. Some of the transgenic mice also developed scabby wounds ontheir shoulders and neck. Skin lesions were observed in a subset of theanimals, generally evolving on areas of skin where hair loss had alreadybeen apparent, and were often exacerbated by the scratching behavior ofthe mice.

RealTime quantitative RT-PCR was used to detect zcytor17lig RNAtranscripts in transgenic (but not non-transgenic) skin samples, withthe Eμ/lck transgenic skin expressing more zcytor17lig RNA than skinfrom EF1α-zcytor17lig transgenic mice. The genes encoding the zcytor17receptor subunits, zcytor17 and OSM-Rbeta, were expressed in the skin ofboth non-transgenic and zcytor17lig-transgenic mice.

An examination of the lymphoid tissues from a subset of theEμ/lck-transgenic founders by flow cytometry revealed a significantincrease in the proportion of activated T cells in the spleen and lymphnodes of these mice. Two of the four mice analyzed had severely enlargedcervical lymph nodes, possibly due to the presence of lesions on theirnecks. A subtle increase in spleen weight and a slight increase inmonocytes and neutrophils circulating in the blood of the transgenicmice was observed. There was no increase in a variety of cytokinestested, nor were there changes in the circulating serum amyloid A levelsin these mice. The effects on the immune cells in the transgenic micemay be a direct or an indirect result of zcytor17lig, or are secondaryeffects of the skin lesions.

Histopathology was performed on many tissues other than skin, includingliver, thymus, spleen, kidney, and testes, and no significantabnormalities in these organs were noted. Analysis of the transgenicskin, however, did reveal a number of alterations, which varied greatlydepending upon the source and location of skin (e.g., normal, hairless,or lesional). In many cases, the ears of the transgenic mice had athickened epidermis as compared to the non-transgenic controls (e.g.,approximately 4 layers versus 2 layers), and the underlying tissuescontained low to moderate numbers of inflammatory cells, which wereprimarily mononuclear with occasional neutrophils. The epidermis overthe abdomen appeared multifocally slightly thicker in the transgenic,but there was no apparent increase in inflammatory cells in theunderlying dermis or subcutis. In the hairless portions of skin fromthese mice, there were dilated hair follicles that contained some debrisbut no hair shafts (e.g., the hairs fell out by the roots). In thelesioned areas, there was severe thickening of the epidermis(acanthosis), increased keratin on the surface of the skin(hyperkeratosis), scattered ulcers of varying size and significantnumbers of inflammatory cells in the dermis (mainly neutrophils, withvarying numbers of macrophages and lymphocytes). The dermis alsocontained numerous mast cells bordering the lesions. Some of the hairshafts in the lesioned areas of the transgenic skin were in the activestage (anagen), in contrast to many of the hair shafts in “normal” areaswhich were in the involuting (catagen) to inactive (telogen) stage.

The phenotype of the zcytor17lig transgenic mice strongly resembles thatof atopic dermatitis (AD) patients, and mouse models of AD. AD is acommon chronic inflammatory disease that is characterized byhyperactivated cytokines of the helper T cell subset 2 (Th2).Zcytor17lig is preferentially expressed by Th2 vs. Th1 cells, whichlends further credence to this comparison. Although the exact etiologyof AD is unknown, multiple factors have been implicated, includinghyperactive Th2 immune responses, autoimmunity, infection, allergens,and genetic predisposition. Key features of the disease include xerosis(dryness of the skin), pruritus (itchiness of the skin), conjunctivitis,inflammatory skin lesions, Staphylococcus aureus infection, elevatedblood eosinophilia, elevation of serum IgE and IgG1, and chronicdermatitis with T cell, mast cell, macrophage and eosinophilinfiltration. Colonization or infection with S. aureus has beenrecognized to exacerbate AD and perpetuate chronicity of this skindisease.

AD is often found in patients with asthma and allergic rhinitis, and isfrequently the initial manifestation of allergic disease. About 20% ofthe population in Western countries suffer from these allergic diseases,and the incidence of AD in developed countries is rising for unknownreasons. AD typically begins in childhood and can often persist throughadolescence into adulthood. Current treatments for AD include topicalcorticosteroids, oral cyclosporin A, non-corticosteroidimmunosuppressants such as tacrolimus (FK506 in ointment form), andinterferon-gamma. Despite the variety of treatments for AD, manypatients' symptoms do not improve, or they have adverse reactions tomedications, requiring the search for other, more effective therapeuticagents.

Epithelial cells, which express the heterodimeric receptor forzcytor17lig (zcytoR17 and OSM-Rbeta), are located at the sites (e.g.,skin, gut, lung, etc.) of allergen entry into the body and interactclosely with dendritic cells (professional antigen presenting cells) insitu. Dendritic cells play an important role in the pathogenesis ofallergic diseases, and it is possible that zcytor17lig can interact withits receptor on epithelial cells in the skin and lung and influenceimmune responses in these organs. Zcytor17lig and its receptor(s) maytherefore contribute to the pathogenesis of allergic diseases such as ADand asthma. Furthermore, the phenotype of the zcytor17lig transgenicmice suggests that this ligand may play a role in wound healing, sincethe mice seem unable to repair damage to their ears, and often bearlong-lasting lesions on their backs and sides. An antagonist ofzcytor17lig might therefore represent a viable therapeutic for these andother indications.

Example 42 Luciferase Assay on Human Transformed Epithelial Cell Linesvia Transient Infection with an Adenoviral STAT/SRE Reporter Gene

A wide variety of human transformed epithelial cell lines (see Table 16below) were seeded in 96-well flat-bottom plates at 10,000 cell/well inregular growth media as specified for each cell type. The following day,the cells were infected with an adenovirus reporter construct, KZ136, ata multiplicity of infection of 5000. The KZ136 reporter contains theSTAT elements in addition to a serum response element. The total volumewas 100 ul/well using DMEM supplemented with 2 mM L-glutamine(GibcoBRL), 1 mM Sodium Pyruvate (GibcoBRL) and 1×Insulin-Transferrin-Selenium supplement (GibcoBRL) (hereinafter referredto as serum-free media). Cells were cultured overnight.

The following day, the media was removed and replaced with 100 μl ofinduction media. The induction media was human zcytor17 ligand dilutedin serum-free media at 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25ng/ml, 3.125 ng/ml and 1.56 ng/ml. A positive control of 20% FBS wasused to validate the assay and to ensure the infection by adenovirus wassuccessful. The cells were induced for 5 hours at which time the mediawas aspirated. The cells were then washed in 50 μl/well of PBS, andsubsequently lysed in 30 μl/well of 1× cell lysis buffer (Promega).After a 10-minute incubation at room temperature, 25 μl/well of lysatewas transferred to opaque white 96-well plates. The plates were thenread on the Luminometer using 5-second integration with 40 μl/wellinjection of luciferase substrate (Promega).

The results revealed the ability of multiple epithelial cell lines torespond to zcytor17 ligand as shown in Table 16 below. TABLE 16 CellLine Species Tissue Morphology Disease Fold Induction A549 Human LungEpithelial Carcinoma 2x Sk-Lu-1 Human Lung Epithelial Adenocarcinoma 6xWI-38 Human Embryonic Lung Fibroblast Negative MRC-5 Human LungFibroblast Negative DU 145 Human Prostate Epithelial Carcinoma 10x PZ-HPV-7 Human Prostate Epithelial Transformed with HPV 5x PC-3 HumanProstate Epithelial Adenocarcinoma Negative U2OS Human Bone EpithelialOsteosarcoma 15.5x   SaOS2 Human Bone Epithelial Osteosarcoma 22x  MG-63Human Bone Fibroblast Osteosarcoma Negative 143B Human Bone FibroblastOsteosarcoma 3.5x   HOS Human Bone Fibroblast and Epithelial 8x TRBMeCHuman Vascular Bone Marrow Epithelial 2x HT144 Human Skin FibroblastMelanoma 5x C32 Human Skin Melanoma Negative Sk-Mel-2 Human SkinPolygonal Melanoma 2.7x   WM-115 Human Skin Epithelial Melanoma 2xHCT-116 Human Colon Epithelial Carcinoma Negative HT-29 Human ColonEpithelial Carcinoma Negative CaCo2 Human Colon EpithelialAdenocarcinoma 3x HBL-100 Human Breast Epithelial 1.5x   ME-180 HumanCervix Epithelial Carcinoma Negative HeLa 299 Human Cervix EpithelialAdenocarcinoma Negative SK-N-SH Human Brain Epithelial NeuroblastomaNegative U138 MG Human Brain Polygonal Glioblastoma Negative HepG2 HumanLiver Epithelial Carcinoma Negative Chang liver Human Liver EpithelialNegative Sk-Hep-1 Human Liver Epithelial Adenocarcinoma 4x Int 407 HumanIntestine Epithelial Negative 3a-Sub E Human Placenta Negative

Example 43 Cytokine Production by Human Epithelial Cell Lines Culturedwith Human Zcytor17 Ligand

Human disease-state epithelial cell lines (A549, human lung epithelialcarcinoma; SkLu1, human lung epithelial adenocarcinoma; DU145, humanprostate epithelial carcinoma; PZ-HPV-7, human prostate epithelial HPVtransformed; U20S, human bone epithelial osteosarcoma) were screened forcytokine production in response to zcytor17 ligand in vitro. These celllines have both zcytor17 and OSMR-beta, identified by RT-PCR, andrespond to human zcytor17 ligand when assayed with the adenoviralluciferase reporter construct, KZ136 (Example 42). Cytokine productionby these cell lines was determined in response to human zcytor17 ligandin a series of three experiments.

A. Cytokine Production by Human Disease-State Epithelial Cell LinesCultured with Human Zcytor17lig

Cells were plated at a density of 4.5×10⁵ cells per well in a 6 wellplate (Costar) and cultured in respective growth media. The cells werecultured with test reagents; 100 ng/mL zcytor17 ligand, 10 ng/mLInterferon gamma (IFN gamma) (R&D Systems, Minneapolis, Minn.), 10 ng/mLTumor Necrosis Factor alpha (TNF alpha) (R&D Systems, Minneapolis,Minn.), 10 ng/mL IL-1beta (R&D Systems, Minneapolis, Minn.) or 100 ug/mLLipopolysaccharide (LPS) (Sigma). Supernatants were harvested at 24 and48 hours and assayed for cytokines; GM-CSF (Granulocyte-MacrophageColony-Stimulating Factor), IL-1b, IL-6, IL-8, MCP-1 (MacrophageChemoattractant Protein-1) and TNFa. Multiplex Antibody Bead kits fromBioSource International (Camarillo, Calif.) were used to measurecytokines in samples. Assays were read on a Luminex-100 instrument(Luminex, Austin, Tex.) and data was analyzed using MasterPlex software(MiraiBio, Alameda, Calif.). Cytokine production (pg/mL) for each cellline in the 24-hour samples is shown below in Table 17. TABLE 17 A549SkLu1 DU145 U2OS PZ-HPV-7 GM-CSF pg/mL zcytor17L 18.80 10.26 16.19 13.2614.10 IFN-g 16.19 13.36 11.56 16.26 11.81 IL-1b 104.60 126.44 76.77338.25 27.32 TNFa 106.67 33.20 58.50 107.09 33.79 LPS 17.64 10.62 11.8125.47 18.34 control 14.81 8.56 13.26 21.67 13.96 IL-1b pg/mL zcytor17L26.90 30.17 28.77 29.07 28.00 IFN-g 29.07 35.33 21.96 26.90 26.73 IL-1b1332.88 1256.17 979.02 1107.35 998.60 TNFa 31.11 33.28 35.33 31.24 25.66LPS 33.28 28.77 29.07 31.11 31.24 control 28.77 28.77 26.73 31.24 29.07IL-6 pg/mL zcytor17L 20.09 26.89 193.05 19.37 17.30 IFN-g 17.52 33.64217.58 27.02 17.63 IL-1b 175.44 5920.19 2375.29 304.08 18.44 TNFa 354.161002.51 1612.17 103.58 18.33 LPS 18.06 35.65 162.18 22.42 17.30 control17.63 27.80 71.23 19.32 17.19 IL-8 pg/mL zcytor17L 86.33 150.81 150.6145.92 6.81 IFN-g 24.07 72.82 163.31 81.78 1.35 IL-1b 1726.24 4083.124407.79 5308.83 124.17 TNFa 3068.68 3811.75 2539.39 3324.02 69.65 LPS20.28 167.13 230.39 115.08 7.95 control 14.92 109.78 107.27 93.44 9.49MCP-1 pg/mL zcytor17L 8.97 187.29 26.84 105.15 7.20 IFN-g 7.30 267.9917.05 88.68 7.71 IL-1b 8.11 8039.84 88.78 3723.81 4.70 TNFa 8.50 7100.37153.26 3826.80 2.80 LPS 9.40 185.83 22.65 61.62 5.61 control 8.16 167.9313.68 47.78 5.61 TNFa pg/mL zcytor17L 16.23 17.52 16.67 15.80 17.09IFN-g 15.80 17.09 15.80 16.65 15.80 IL-1b 16.66 17.09 15.80 17.95 16.23TNFa 1639.92 1648.83 2975.07 1348.33 3554.82 LPS 16.87 15.80 15.37 17.0917.52 control 16.23 15.80 15.80 17.09 16.66

All cell lines tested produced GM-CSF and IL-8 in response tostimulation with control cytokines IL-1b and TNFa. Most cell linesproduced IL-6 and MCP-1in response to IL-1b and TNFa stimulation.Zcytor17 ligand stimulated IL-6 production in the DU145 cell linecompared to control (193 pg/mL vs. 71 pg/mL). Zcytor17 ligand stimulated3 of 5 cell lines to produce IL-8 with the greatest effect seen in A549cells (5 fold), and reduced IL-8 production in U2OS cells by 2 fold.There was a slight effect on MCP-1 production by DU145 and U2OS cellswhen cultured with zcytor17 ligand.

B. Cytokine Production by Normal Human Epithelial Cell Lines Culturedwith Human Zcytor17lig

In addition to the human epithelial cell lines, normal human bronchialepithelial cells (NHBE, Clonetics) were also tested. Cells were platedat a density of 1×10⁵ cells per well in a 24 well plate and culturedwith test reagents; 1000 ng/mL, 100 ng/mL and 10 ng/mL zcytor17 ligand(A760F), 10 ng/mL TNFa, 10 ng/mL OSM, 10 ng/mL IFNa, 10 ng/mL TGFb or 10ng/mL Lymphotactin. Supernatants were harvested at 24 and 48 hours andassayed for cytokines; IL-6, IL-8, MCP-1, MIP-1a, RANTES and Eotaxin.Cytokines were assayed as previously described. Cytokine production(pg/mL) for each cell line in the 48-hour samples is shown below inTable 18. TABLE 18 A549 DU145 SkLu1 U2OS NHBE IL-6 pg/ml r17L 1000 ng/ml24.5 56.3 32.1 25.2 64.5 r171L 100 ng/ml 25.0 65.0 31.0 25.4 50.2 r17L10 ng/ml 24.8 51.8 30.2 25.3 54.3 TNFa 272.9 355.4 437.5 36.1 299.3 OSM26.4 73.5 112.4 25.6 80.4 IFNa 24.6 109.3 33.7 26.4 52.4 TGFb 24.4 102.642.7 27.8 268.9 control 24.5 36.3 29.9 25.2 47.9 IL-8 pg/ml r17L 1000ng/ml 35.0 243.3 45.6 18.6 402.0 r171L 100 ng/ml 31.0 290.7 40.1 21.3296.0 r17L 10 ng/ml 30.4 240.4 33.4 18.9 361.8 TNFa 2809.3 2520.9 1385.2784.9 1486.3 OSM 37.8 60.6 68.0 22.5 494.6 IFNa 18.9 315.3 39.5 33.1231.6 TGFb 9.9 77.5 19.6 88.9 246.9 control 10.9 238.0 38.0 39.7 315.8MCP-1 pg/ml r17L 1000 ng/ml nd nd 149.1 81.0 nd r171L 100 ng/ml nd nd130.6 81.9 nd r17L 10 ng/ml nd nd 111.7 49.1 nd TNFa nd 22.1 2862.61104.7 nd OSM nd 17.2 448.2 85.8 nd IFNa nd nd 131.7 10.5 nd TGFb nd 1.754.5 27.6 nd control nd nd 113.0 1.7 ndnd = not detected

DU145 cells produced IL-6 in response to zcytor17 ligand, repeating theprevious results in Example 43A. However, only A549 and U2OS had similarIL-8 responses as seen Example 43A. SkLu1 and U2OS cells both producedMCP-1 in response to zcytor17 ligand. Cytokine production by NHBE cellswas marginal compared to controls.

C. Cytokine Production by Human Disease-State Epithelial Cell LinesCo-Cultured with Human Zcytor17lig and IFN Gamma

Cells were plated at a density of 2×10⁵ cells per well in 24 well plateand co-cultured with 10 ng/mL IFN gamma±zcytor17 ligand at 100 ng/mL, 10ng/mL or 1 ng/mL. Supernatants were collected at 24 and 48 hours andassayed for IL-8 and MCP-1 as described above. Cytokine production(pg/mL) for each cell line in the 24-hour samples is shown below inTable 19. TABLE 19 IL-8 pg/ml MCP-1 pg/ml A549 10 ng/mL IFNg + 100 ng/mLr17L 86.7 nd 10 ng/mL IFNg + 10 ng/mL r17L 75.1 nd 10 ng/mL IFNg + 1ng/mL r17L 63.6 nd 10 ng/ml IFNg 35.4 nd control 36.6 nd DU145 10 ng/mLIFNg + 100 ng/mL r17L 102.3 nd 10 ng/mL IFNg + 10 ng/mL r17L 92.9 nd 10ng/mL IFNg + 1 ng/mL r17L 79.9 nd 10 ng/ml IFNg 70.7 nd control 79.4 ndSkLu1 10 ng/mL IFNg + 100 ng/mL r17L 152.2 604.9 10 ng/mL IFNg + 10ng/mL r17L 194.4 870.7 10 ng/mL IFNg + 1 ng/mL r17L 138.7 585.4 10 ng/mlIFNg 170.8 652.6 control 203.0 292.3 U2OS 10 ng/mL IFNg + 100 ng/mL r17L106.8 357.0 10 ng/mL IFNg + 10 ng/mL r17L 108.2 347.7 10 ng/mL IFNg + 1ng/mL r17L 109.9 293.3 10 ng/ml IFNg 118.8 159.8 control 146.8  7.0

A549 cells produced IL-8 in response to zcytor17 ligand, however therewas no effect of co-culturing cells with the addition of IFN gamma. U2OScells made 20 fold more MCP-1 when cultured with IFNg and 50 fold moreMCP-1 when cultured with IFN gamma+zcytor17 ligand.

Example 44 Zcytor17lig Effects on ³H-TdR Incorporation in DU145 ProstateEpithelial Carcinoma Cells

Cells were seeded in 96-well tissue clusters (Falcon) at a density of25,000/well in MEM (Life Technologies) growth medium supplemented withglutamine, pyruvate, non-essential amino acids (Life Technologies) and10% fetal bovine serum (Hyclone). At confluence (24 hours later), cellswere switched to growth arrest media by substituting 0.1% BSA (LifeTechnologies) for serum. After 48 hours to achieve cell synchronization,the growth-arrest medium was replaced with fresh medium. Then, humanrecombinant zcytor17lig (test reagent) was added at variousconcentrations (from 0.24 to 60 ng/mL) (see Table 16 below), to test forthe effect of the protein on basal DNA replication. Some wells received2.5% FBS (Hyclone) in addition to zcytor17Ligand, in order to testeffect of the protein on elevated levels of TdR incorporation. FBS 10%and 20 ng/ml Platelet Derived Growth Factor-BB (PDGF-BB) (R&D) were usedas positive control.

Eighteen hours following addition of zcytor17Ligand and the rest of thetest reagents, cells were pulsed with 250 nCi/mL [³H]-thymidine (NEN)for 4 hours. Following the 4-hour pulse, media were discarded and 100 μLtrypsin solution (Life Technologies) was added in each well to dislodgethe cells. The radioactivity incorporated by DU145 was determined byharvesting the cells with a Packard Filtermate 196 cell harvester and bycounting the incorporated label using a Packard TopCount NXT microplatescintillation counter.

As can be seen in Table 20 below, zcytor17lig induced thymidineincorporation in quiescent cells (in 0.1% BSA) in aconcentration-dependent manner. This effect reached 2.5-fold of the BSAcontrol at the highest concentration used, 60 ng/mL. In addition, thiseffect of zcytor17lig was also detectable when the baselineincorporation was elevated by the addition of 2.5% FBS (in this seriesas potent a mitogen as 10% FBS). These results therefore indicate thatunder both basal and stimulated conditions zcytor17lig can act as amitogenic factor for the DU145 carcinoma cells.

Table 16 shows the effects of zcytor17lig on thymidine incorporation byDU145 cells. Results are expressed in cpm/well and numbers are themean±st.dev of triplicate wells. TABLE 20 0.1% BSA 2.5% FBS BSA Control1139 ± 336 4228 ± 600 Zcytor17lig (0.24 ng/mL) 1430 ± 136  4894 ± 1037Zcytor17lig (0.74 ng/mL) 1657 ± 32  5038 ± 810 Zcytor17lig (2.22 ng/mL)1646 ± 57  5162 ± 808 Zcytor17lig (6.67 ng/mL) 2226 ± 189  6385 ± 1613Zcytor17lig (20 ng/mL) 2168 ± 108  5880 ± 1085 Zcytor17lig (60 ng/mL)2512 ± 111 6165 ± 417 PDGF-BB (20 ng/mL) 4094 ± 202 5927 ± 360

Example 45 Expression of Huzcytor17Ligand in E. coli

A. Construction of Expression Vector pRPS01 that ExpressesHuzcytor17Lig/MBP-6H Fusion Polypeptide

An expression plasmid containing a polynucleotide encoding ahuzcytor17lig fused C-terminally to Maltose Binding Protein (MBP) wasconstructed via homologous recombination. The fusion polypeptidecontains an N-terminal approximately 388 amino acid MBP portion fused tothe huzcytor17Lig described herein. A fragment of huzcytor17lig cDNA wasisolated using the PCR method as described herein. Two primers were usedin the production of the zcytor17lig fragment in a standard PCRreaction: (1) one containing 40 bp of the vector flanking sequence and20 bp corresponding to the amino terminus of the huzcytor17lig, and (2)another containing 40 bp of the 3′ end corresponding to the flankingvector sequence and 20 bp corresponding to the carboxyl terminus of thehuzcytor17lig. Two microliters of the 100 μl PCR reaction was run on a1.0% agarose gel with 1×TBE buffer for analysis, and the expectedmolecular weight fragment was observed. The remaining PCR reaction wascombined with the second PCR tube and precipitated with 400 μl ofabsolute ethanol. The precipitated DNA was used for recombination intothe Sma1 cut recipient vector pTAP98 to produce the construct encodingthe MBP-huzcytor17lig fusion, as described below.

The vector pTAP98 was constructed using yeast homologous recombination.One hundred nanograms of EcoR1 cut pMAL-c2 was recombined with 1 μg Pvu1cut pRS316, 1 μg linker, and 1 g Sca1/EcoR1 cut pRS316 were combined ina PCR reaction. PCR products were concentrated via 100% ethanolprecipitation. The competent yeast cell (S. cerevisiae) strain,SF838-9Dα, was combined with 10 μl of a mixture containing approximately1 μg of the huzcytor17lig PCR product (above) and 100 ng of SmaIdigested pTAP98 vector, and electroporated at 0.75 kV, 25 μF and ∞ ohms.The resulting reaction mixture was plated onto URA-D plates andincubated at 30° C.

After 48 hours, the Ura+ yeast transformants from a single plate wereselected. DNA was isolated and transformed into electrocompetent E. colicells (e.g., MC1061, Casadaban et. al. J. Mol. Biol. 138, 179-207). Theresulting E. coli cells were plated on MM/CA+AMP 100 mg/L plates (Pryorand Leiting, Protein Expression and Purification 10:309-319, 1997) usingstandard procedures. Four individual clones were harvested from theplates and inoculated into MM/CA with 100 μg/ml Ampicillin for two hoursat 37° C. One milliliter of each of the culture was induced with 1 mMIPTG. Approximately 2-4 hours later, 250 μl of each induced culture wasmixed with 250 μl acid washed glass beads and 250 μl Thorner buffer with5% βME and dye (8M urea, 100 mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5%SDS). Samples were vortexed for one minute and heated to 65° C. for 10minutes. Twenty microliters of each sample was loaded per lane on a4%-12% PAGE gel (OVEX). Gels were run in 1×MES buffer. The positiveclones were designated pRPS01 and subjected to sequence analysis.

One microliter of sequencing DNA was used to transform electrocompetentE. coli cell strain MC1061. The cells were electropulsed at 2.0 kV, 25μF and 400 ohms. Following electroporation, cells were rescued 0.6 mlSOC and grown on LB+Amp plates at 37° C. overnight, with 100 mg/LAmpicillin. Four cultures were induced with ITPG and screened forpositives as described above. The positive clones were expanded forprotein purification of the huzcytor17lig/MBP-6H fusion protein usingstandard techniques.

B. Purification of Huzcytor17Lig/MBP-6H from E. coli Fermentation

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used to purify recombinant huzcytor17Lig/MBP-6Hpolypeptide. E. coli cells containing the pRPS01 construct andexpressing huzcytor17Lig/MBP-6H, were constructed using standardmolecular biology methods and cultured in 50.0 g/L SuperBroth II (12 g/LCasien, 24 g/L Yeast Extract, 11.4 g/L di-potassium phosphate, 1.7 g/LMono-potassium phosphate; Becton Dickenson, Cockeysville, Md.), 5 g/Lglycerol and 5 mL/L 1M Magnesium Sulfate. Twenty grams of cells wereharvested and frozen for protein purification.

The thawed cells were resuspended in 500 mL Amylose Equilibration buffer(20 mM Tris, 100 mM NaCl, pH 8.0). A French Press cell breaking system(Constant Systems Ltd., Warwick, UK) with a temperature setting of −7°C. to −10° C. and 30K PSI was used to lyse the cells. The resuspendedcells were assayed for breakage by A₆₀₀ readings before and aftercycling through the French Press. The processed cell suspension waspelleted at 10,000 G for 30 minutes to remove the cellular debris andthe supernatant was harvested for protein purification.

A 25 ml column of Amylose resin (New England Biolabs, Beverly, Mass.)(prepared as described below) was poured into a Bio-Rad, 2.5 cm D×10 cmH glass column. The column was packed and equilibrated by gravity with10 column volumes (CVs) of Amylose Equilibration buffer. The processedcell supernatant was batch loaded to the Amylose resin overnight, withrocking. The resin was returned to the Bio-Rad column and washed with 10CV's of Amylose Equilibration buffer by gravity. The column was elutedwith ˜2 CV of Amylose Elution buffer (Amylose Equilibration buffer+10 mMMaltose, Fluka Biochemical, Switzerland) by gravity. Ten 5 mL fractionswere collected over the elution profile and assayed for Absorbance at280 and 320 nM. The Amylose resin was regenerated with 1 CV of distilledH₂O, 5 CVs of 0.1% (w/v) SDS (Sigma), 5 CVs of distilled H₂O, 5 CVs ofAmylose Equilibration buffer and finally 1 CV of Amylose Storage buffer(Amylose Equilibration buffer+0.02% Sodium Azide). The regeneratedcolumn was stored at 4° C.

Elution profile fractions of interest were pooled and dialyzed in a 10Kdialysis chamber (Slide-A-Lyzer, Pierce Immunochemicals) against 4×4 LPBS pH 7.4 (Sigma) over an 8 hour time period to remove low molecularweight contaminants, buffer exchange and desalt. Following dialysis, thematerial harvested represented the purified huzcytor17Lig/MBP-6Hpolypeptide. The purified huzcytor17Lig/MBP-6H polypeptide was filtersterilized and analyzed via SDS-PAGE Coomassie staining for anappropriate molecular weight product. The concentration of thehuzcytor17Lig/MBP-6H polypeptide was determined by BCA analysis to be1.28 mg/mL.

Example 46 Human Zcytor17lig Polyclonal Antibody

A. Preparation and Purification

Polyclonal antibodies were prepared by immunizing 2 female New Zealandwhite rabbits with the purified recombinant protein hzcytor17L/MBP-6H(Example 45). The rabbits were each given an initial intraperitoneal(IP) injection of 200 μg of purified protein in Complete Freund'sAdjuvant followed by booster IP injections of 100 μg protein inIncomplete Freund's Adjuvant every three weeks. Seven to ten days afterthe administration of the second booster injection (3 total injections),the animals were bled and the serum was collected. The animals were thenboosted and bled every three weeks.

The hzcytor17L/MBP-6H specific rabbit serum was pre-adsorbed of anti-MBPantibodies using a CNBr-SEPHAROSE 4B protein column (Pharmacia LKB) thatwas prepared using 10 mg of non-specific purified recombinant MBP-fusionprotein per gram of CNBr-SEPHAROSE. The hzcytor17L/MBP-6H-specificpolyclonal antibodies were affinity purified from the pre-adsorbedrabbit serum using a CNBr-SEPHAROSE 4B protein column (Pharmacia LKB)that was prepared using 10 mg of the specific antigen purifiedrecombinant protein hzcytor17L/MBP-6H. Following purification, thepolyclonal antibodies were dialyzed with 4 changes of 20 times theantibody volume of PBS over a time period of at least 8 hours.Hzcytor17-Ligand-specific antibodies were characterized by ELISA using500 ng/ml of the purified recombinant proteins hzcytor17L/MBP-6H orhzcytor17L-CEE produced in a baculovirus expression system as antibodytargets. The lower limit of detection (LLD) of the rabbitanti-hzcytor17L/MBP-6H affinity purified antibody is 100 μg/ml on itsspecific purified recombinant antigen hzcytor17L/MBP-6H and 500 μg/ml onpurified recombinant hzcytor17L-CEE produced in a baculovirus expressionsystem.

B. SDS-PAGE and Western Blotting Analysis of Rabbit Anti-HumanZcytoR17lig MBP-6H Antibody

Rabbit Anti-human ZcytoR17lig MBP-6H antibody was tested by SDS-PAGE(NuPage 4-12%, Invitrogen, Carlsbad, Calif.) with coomassie stainingmethod and Western blotting using goat anti-rabbit IgG-HRP. Human andmouse zcytor17lig purified protein (200-25 ng) was electrophoresed usingan Invitrogen Novex's Xcell II mini-cell, and transferred tonitrocellulose (0.2 mm; Invitrogen, Carlsbad, Calif.) at roomtemperature using Novex's Xcell blot module with stirring according todirections provided in the instrument manual. The transfer was run at300 mA for one hour in a buffer containing 25 mM Tris base, 200 mMglycine, and 20% methanol. The filter was then blocked with Western Abuffer (in house, 50 mM Tris, pH 7.4, 5 mM EDTA, pH 8.0, 0.05% IgepalCA-630, 150 mM NaCl, and 0.25% gelatin) overnight with gentle rocking at4° C. The nitrocellulose was quickly rinsed, then the rabbit anti-humanzcytoR17lig MBP-6H (1:1000) was added in Western A buffer. The blot wasincubated for 1.5 hours at room temperature with gentle rocking. Theblot was rinsed 3 times for 5 minutes each in Western A, then goatanti-rabbit IgG HRP antibody (1:5000) was added in Western A buffer. Theblot was incubated for 1 hour at room temperature with gentle rocking.The blot was rinsed 3 times for 5 minutes each in Western A, thenquickly rinsed in H₂O. The blot was developed using commerciallyavailable chemiluminescent substrate reagents (ECL Western blottingdetection reagents 1 and 2 mixed 1:1; reagents obtained from AmershamPharmacia Biotech, Buckinghamshire, England) and the blot was exposed tox-ray film for up to 5 minutes.

The purified human zcytor17lig appeared as a large band at about 30 kDaand a smaller band at about 20 kDa under reduced conditions. The mousezcytor17lig was not detected by the rabbit anti-human zcytor17ligantibody.

Example 47 Zcytor17lig Effects on U93 7 Monocyte Adhesion to TransformedBone Marrow Endothelial Cell (TRBMEC) Monolayer

Transformed Bone Marrow Endothelial Cells (TRBMEC) were seeded in96-well tissue clusters (Falcon) at a density of 25,000/well in mediumM131 (Cascade Biologics) supplemented with Microvascular GrowthSupplement (MVGS) (Cascade Biologics). At confluence (24 hours later),cells were switched to M199 (Gibco-Life Technologies) supplemented with1% Fetal Bovine Serum (Hyclone). Human recombinant zcytor17lig (testreagent) was added at various concentrations (from 0.4 to 10 ng/mL) (seeTable 21 below), to test for the effect of the protein on immunecell-endothelial cell interactions resulting in adhesion. Some wellsreceived 0.3 ng/ml Tumor Necrosis Factor (TNFalpha R&D Systems), a knownpro-inflammatory cytokine, in addition to zcytor17lig, to test an effectof the protein on endothelial cells under inflammatory conditions.TNFalpha at 0.3 ng/ml alone was used as positive control and theconcentration used represents approximately 70% of the maximal TNFalphaeffect in this system, i.e., it does not induce maximal adherence ofU937 cells (a human monocyte-like cell line) to the endothelium. Forthis reason, this setup can detect both upregulation and downregulationof the TNFalpha effects. Basal levels of adhesion both with and withoutTNFalpha were used as baseline to assess effect of test reagents.

After overnight incubation of the endothelial cells with the testreagents (zcytor17ligand±TNFalpha), U937 cells, stained with 5 μMCalcein-AM fluorescent marker (Molecular Probes), the cells weresuspended in RPMI 1640 (no phenol-red) supplemented with 1% FBS andplated at 100,000 cells/well on the rinsed TRBMEC monolayer.Fluorescence levels at excitation/emission wavelengths of 485/538 nm(Molecular Devices micro-plate reader, CytoFluor application) weremeasured 30 minutes later, before and after rinsing the well three timeswith warm RPMI 1640 (no phenol-red), to remove non-adherent U937.Pre-rinse (total) and post-rinse (adherence-specific) fluorescencelevels were used to determine percent adherence (net adherent/nettotal×100=% adherence).

As can be seen in Table 21 below, zcytor17lig when added alone affectedthe basal adherence of U937 cells to the endothelial monolayers at theconcentration range used (less than 2-fold increases, p<0.01 by ANOVAtest). By itself, the positive control, 0.3 ng/mL TNFalpha, increasedthe adherence of U937 cells from a basal 5.8% to 35% (6-fold). In thepresence of TNFalpha, zcytor17lig synergized with TNFalpha and furtherenhanced U937 adhesion in a concentration-dependent manner between 0.4and 10 ng/mL (p<0.01 by ANOVA test). At 10 ng/mL, zcytor17lig enhancedthe effect of TNFalpha by 62%. These results indicate that zcytor17ligmay by itself be a pro-inflammatory agent. Zcytor17lig was able tosynergize with sub-maximal concentrations of TNFalpha to increasemonocyte adherence to endothelial cells. These results also show thatendothelial cells, especially when exposed to pro-inflammatory cytokinessuch as TNFalpha, are a likely target tissue of zcytor17lig action. Theconsequence of zcytor17ligand on endothelial cells may be to heightenmonocyte or macrophage adhesion to a site of proinflammatory activity.Activated monocytes and macrophages are important in many inflammatorydiseases. Therefore inhibition of monocyte/macrophage adhesions mayprovide a therapeutic rationale for zcytor17ligand antagonists. Thisdata would support the use of zcytor17 ligand antagonists for treatmentlung diseases, vascular diseases, autoimmunity, tumor metastasis,disease involving allergic reactions, wound healing and diseases of theskin including contact, allergic or non-allergic dermatistic orpsoriasis and inflammatory bowel disease. Table 21 shows the effects ofzcytor17lig on U937 monocyte adhesion to TRBMEC endothelial monolayers.Results are expressed in percent adhesion and numbers are themean±st.dev of triplicate wells. TABLE 21 Basal 0.3 ng/mL TNFalpha Basal5.8 ± 1.2   35 ± 5.5 zcytor17lig 0.4 ng/mL   9 ± 0.7 44.7 ± 2.5zcytor17lig 1.1 ng/mL 10.4 ± 0.8  45.2 ± 0.6 zcytor17lig 3.3 ng/mL 7.9 ±1.7 51.1 ± 4   zcytor17lig 10 ng/mL 9.5 ± 0.5 56.6 ± 3.9

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated receptor polypeptide comprising an amino acid sequencehaving at least 95% sequence identity with amino acid residues 20-732 ofSEQ ID NO:111, wherein the receptor polypeptide forms a heterodimeric ormultimeric receptor polypeptide with a second receptor polypeptidecomprising amino acid residues 28-979 of SEQ ID NO:7, and wherein theheterodimeric or multimeric receptor polypeptide binds a ligandcomprising amino acid residues 27-164 of SEQ ID NO:2.
 2. The isolatedreceptor polypeptide of claim 1 wherein the receptor polypeptidecomprises amino acid residues 20-732 of SEQ ID NO:111.
 3. The isolatedreceptor polypeptide of claim 1 wherein the second receptor polypeptidecomprises amino acid residues 1-732 of SEQ ID NO:7.
 4. An isolatedsoluble receptor polypeptide comprising an amino acid sequence having atleast 95% sequence identity with amino acid residues 20-519 of SEQ IDNO:111, wherein the soluble receptor polypeptide forms a solubleheterodimeric or multimeric receptor polypeptide with a second solublereceptor polypeptide comprising amino acid residues 28-739 of SEQ IDNO:7, and wherein the soluble heterodimeric or multimeric receptor bindsa ligand comprising amino acid residues 27-164 of SEQ ID NO:2.
 5. Theisolated soluble receptor polypeptide of claim 4 wherein the receptorpolypeptide comprises amino acid residues 20-732 of SEQ ID NO:111. 6.The isolated soluble receptor polypeptide of claim 4 wherein the solubleheterodimeric or multimeric receptor polypeptide antagonizesinflammation induced by said ligand.
 7. The isolated soluble receptorpolypeptide of claim 4 wherein the soluble heterodimeric or multimericreceptor further comprises an immunoglobulin heavy chain constantregion.
 8. The isolated soluble receptor polypeptide of claim 7 whereinthe immunoglobulin heavy chain constant region is an F_(c) fragment. 9.A composition comprising: a soluble receptor polypeptide comprising anamino acid sequence having at least 95% sequence identity with aminoacid residues 20-519 of SEQ ID NO:111, wherein the soluble receptorpolypeptide forms a soluble heterodimeric or multimeric receptorpolypeptide with a second soluble receptor polypeptide comprising aminoacid residues 28-739 of SEQ ID NO:7; and a pharmaceutically acceptablevehicle.
 10. The composition of claim 9 wherein the solubleheterodimeric or multimeric receptor polypeptide is fused to animmunoglobulin heavy chain constant region.
 11. The composition of claim10 wherein the immunoglobulin heavy chain constant region is an F_(c)fragment.